ABL TYROSINE KINASES MEDIATE INTERCELLULAR ADHESION. Nicole L. Zandy. Department of Pharmacology and Cancer Biology Duke University.

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1 ABL TYROSINE KINASES MEDIATE INTERCELLULAR ADHESION by Nicole L. Zandy Department of Pharmacology and Cancer Biology Duke University Date: Approved: Ann Marie Pendergast, Supervisor Christopher Counter Mark Dewhirst Christopher Kontos Xiao Fan Wang Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2008

2 ABSTRACT ABL TYROSINE KINASES MEDIATE INTERCELLULAR ADHESION by Nicole L. Zandy Department of Pharmacology and Cancer Biology Duke University Date: Approved: Ann Marie Pendergast, Supervisor Christopher Counter Mark Dewhirst Christopher Kontos Xiao Fan Wang An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2008

3 Copyright by Nicole Lynn Zandy 2008

4 Abstract Adherens junctions are calcium dependent cell cell contacts formed during epithelial morphogenesis that link neighboring cells via cadherin receptors. Coordinated regulation of the actin cytoskeleton by the Rho GTPases is required for the formation and dissolution of adherens junctions, however the pathways that link cadherin signaling to cytoskeletal regulation remain poorly defined. The Abl tyrosine kinases been shown to modulate cytoskeletal reorganization downstream of various extracellular signals including growth factor receptors and integrins. Here we use pharmacological inhibition, genetic inactivation, and RNA interference to identify the Abl family kinases as critical mediators of cadherin mediated adhesion. Endogenous Abl family kinases, Abl and Arg, are activated and are required for Rac activation following cadherin engagement, and regulate the formation and maintenance of adherens junctions in mammalian cells. Significantly, we show that Abldependent regulation of the Rho ROCK myosin signaling pathway is critical for the maintenance of adherens junctions. Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its downstream target ROCK, leading to enhanced phosphorylation of the myosin regulatory light chain. These signaling events result in enhanced stress fiber formation and increased acto myosin contractility, thereby disrupting adherens junctions. Conversely, Arg gain of function promotes adherens iv

5 junction formation through a Crk dependent pathway in cells with weak junctions. These data identify the Abl kinases as a novel regulatory link between the cadherin/catenin adhesion complex and the actin cytoskeleton through regulation of Rac and Rho during adherens junction formation. Unexpectedly, we identified a requirement for Abl and Crk downstream of Rac in the regulation of adherens junctions. Therefore, Abl functions both upstream and downstream of Rac in regulating adherens junctions, which suggests the possibility of a positive feedback loop consisting of Abl Crk Rac. Finally, we identified the Abl kinases as critical mediators of epithelial cell response to HGF. Pharmacological inhibition of Abl kinase activity resulted in impaired dissolution of adherens junctions downstream of HGF stimulation of the Met receptor. Additionally, we observed decreased phosphorylation of the Met receptor itself, along with Gab1 and Crk, downstream effectors of Met signaling. Taken together, these data suggest a requirement for Abl kinases in both adherens junctions formation and turnover. v

6 Dedication It is with a sense of profound loss and utter desolation that I dedicate this dissertation in loving memory of my father, Leonard Marino Zandy (Sunday, August 6, 1944 Friday, November 23, 2007). Exactly five months have passed since I lost my first valentine, my career role model, my sports buddy and athletic trainer, my TV gossip companion, my project manager, my verbal sparring partner, my first styling client, my inspiration to achieve without costing others and to give until it hurts, the foundation of my moral and ethical beliefs, the sun in my solar system, the center of my family, and the platinum standard I use to gauge the value of husband and father. Just two days and five months ago, I was exhausted, in part due to our trip to South Bend, but happily submitting my dissertation in plenty of time to finish dinner before watching Duke win the Maui Invitational and looking forward to seeing Santa Claus in the Macy s Thanksgiving Day Parade the next day. On Thanksgiving Day, my dad, mom, Kim, Pam, and I sat at the table eating turkey, roast beast, and mashed potatoes, while Kurbain ate a cornucopia of food donations. It was a perfect meal. On Friday, I woke up with a headache, but insisted I would not miss our family tradition of me & mommy & daddy cutting down our Christmas tree I had to get it decorated before I left to return to Durham to prepare for my thesis defense the vi

7 following week. We all got ready, went to our usual local tree farm, and almost too easily found the perfect tree, our first Fraser Fir from PA in years, and brought it home. The events that followed are too private to be shared: and just like that he was gone And in that instant, I learned infinitely more than the words that follow on these hundred odd pages describing five years work convey. It is the wisdom from that moment that I will continue to build on and share in his name. I miss you, Daddy. xoxo Love, Nicole vii

8 Contents Abstract...iv Dedication...vi List of Figures...xii 1. Introduction Adherens junctions Adherens junctions during development Adherens junctions in cancer Adherens junction components Cadherin family proteins Catenin family proteins Actin cytoskeleton Regulation of adherens junctions Adherens junction component binding Phosphorylation Rho GTPases Cross talk between Adherens junctions and Receptor Tyrosine Kinases Interactions between adherens junctions and RTKs Met receptor tyrosine kinase Cancer relevance Biological effects of Met activation...30 viii

9 Met signaling Abl family of non receptor Tyrosine Kinases Abl and Arg Regulation of Abl/Arg Physical Protein modification Upstream signals regulating Abl tyrosine kinase activity Biological function of Abl/Arg In vivo roles of Abl and Arg Abl kinases regulate response to growth factors and cell migration Abl family kinases in intercellular conversations Abl Tyrosine Kinases regulate cell cell adhesion via Rho GTPAses Introduction Results Abl Tyrosine Kinases are Required for Cell Cell Adhesion Abl Tyrosine Kinases are Required for the Maintenance of Adherens Junctions Cell cell Adhesion Leads to Abl Kinase Activation and Recruitment to Sites of Cell Cell Contact Abl Kinases Regulate Rac Activation in Response to Cadherin Engagement via Crk/CrkL Abl Kinases Regulate the Architecture of the Actin Cytoskeleton in Epithelial Cell Sheets via the Rho Rock Pathway...73 ix

10 2.3 Discussion A closer look at Abl Crk Rac Background Results Discussion Role for Abl kinases downstream of the Met receptor Background Results Abl kinases function downstream of the activated Met receptor tyrosine kinase for induction of cell scattering Abl kinase inhibition alters signaling downstream of the activated Met receptor Discussion Materials and Methods Antibodies and Reagents Cell Lines Retroviral Transduction Cell lysis, immunoblotting, in vitro kinase assays, and RhoGTPase activation assays Immunofluorescence Calcium Switch Experiments sirna Growth factor stimulation x

11 6. Conclusion Delving deeper into signaling downstream of cadherins Role for Abl and Crk Abl and p190 RhoGAP Abl and other downstream effectors Dissecting the role of Abl downstream of Met receptor activation Abl and Met Abl and other downstream targets Role of Abl downstream of other growth factors Abl and FGF Abl and VEGF Concluding remarks References Biography xi

12 List of Figures Figure 1: Schematic representation of adherens junctions...3 Figure 2: Functional domains of c Abl and Arg Figure 3: Regulation of Abl kinase activity Figure 4: Loss of Abl family kinases in fibroblasts disrupts N cadherin based adhesion Figure 5: Inhibition of Abl kinase activity or protein impairs adherens junction formation in epithelial cells...60 Figure 6: Loss of Abl/Arg impairs intercellular adhesion...61 Figure 7: Inhibition of Abl kinase activity impairs accumulation of E cadherin catenin complexes at cell cell contacts...63 Figure 8: Cell cell adhesion regulates the activity of endogenous Abl kinases Figure 9: Arg localizes to adherens junctions Figure 10: Crk/CrkL function downstream of Abl mediated adherens junction formation in epithelial cells...70 Figure 11: Abl kinases regulate Rac activation in response to cadherin engagement...72 Figure 12: Abl kinases regulate the actin cytoskeleton and Rho activity in epithelial cell monolayers...74 Figure 13: Disruption of adherens junctions in response to inhibition of Abl kinase activity is reversed by inhibition of ROCK kinase activity Figure 14: Model for Abl family kinase regulation of adherens junctions Figure 15: Abl and Crk function downstream of Rac activation...87 xii

13 Figure 16: ArgPP expression rescues negative effects of Crk overexpression on adherens junctions Figure 17: Proposed model for Abl Crk Rac signaling Figure 18: Hyperactivation of Abl kinase activity in metastatic breast cancer cell lines..95 Figure 19: Abl kinases act downstream of c Met following HGF stimulation...99 Figure 20: Abl kinase inhibition blocks tyrosine phosphorylation of Crk in response to Met activation Figure 21: Inhibition of Abl kinase activity affects Met signaling downstream of HGF stimulation Figure 22: Lack of evidence for role of Abl kinase modulation of p190 RhoGAP effect on adherens junctions Figure 23: FGF2 stimulation activates endogenous Abl kinases Figure 24: Abl kinases mediate intercellular adhesion in endothelial cells xiii

14 1. Introduction 1.1 Adherens junctions Adherens junctions are one of several types of specialized structures, which allow cells to organize into tissues. Found at the basolateral surface connecting two neighboring cells, they may line the contacts between cells continuously (as in epithelial cells) or are arranged in a more discontinuous fashion (as in fibroblasts). In addition to promoting intercellular adhesion, adherens junctions promote the establishment of apical basal polarity by providing a loose physical barrier and are required for the formation of tight junctions (Vleminckx and Kemler, 1999) Adherens junctions during development Intercellular adhesion relies on the function and interaction of many cell adhesion molecules, including classical cadherins, integrins, immunoglobulin type adhesion molecules (Ig CAMs), and distinct pairs of membrane bound ligand/receptors such as ephrins/eph and semaphorins/plexins (Comoglio et al., 2004; Thiery, 2002; Vleminckx and Kemler, 1999). The engagement of adhesion receptors leads to activation of downstream signaling pathways resulting in dynamic remodeling of the cytoskeleton, which is critical for their adhesive properties. Dynamic regulation of the actin cytoskeleton is required for the formation and dissolution of intercellular adhesions 1

15 during tissue morphogenesis, cell migration, differentiation during gastrulation, synaptogenesis, wound healing, and pathological processes such as tumor invasion and metastasis (Thiery, 2002; Vasioukhin and Fuchs, 2001). Formation of cell cell contacts in multicellular organisms is critically dependent on adherens junctions (Schock and Perrimon, 2002). Impaired formation of adherens junctions is associated with developmental defects associated with loss of adhesion (Cavallaro and Christofori, 2004; Thiery, 2002). Adherens junctions are specialized structures formed during epithelial morphogenesis which physically link the actin cytoskeletons of neighboring cells through cadherin molecules, which bind to one another in a calcium dependent manner (Schock and Perrimon, 2002) (Figure 1). The extracellular domains of the cadherins dimerize and cluster, which triggers the indirect association of cadherin cytoplasmic tails with the actin cytoskeleton. This indirect link is mediated by the binding of the cadherin tail to β catenin, which in turn binds α catenin, which ultimately links the cadherin/catenin complex to the actin cytoskeleton (Kobielak and Fuchs, 2004). Epithelial (E) cadherin and the catenins play a critical role in promoting normal epithelial morphogenesis. Their importance is demonstrated by the early embryonic lethality of mice that carry null mutations of the corresponding genes (Jamora and Fuchs, 2002). E cadherin expression is important for maintaining epithelial morphology, 2

16 P120 catenin α β α - catenin β - catenin P120 catenin Cadherin P120 cat. β α β - cat. α - cat. F Actin Figure 1: Schematic representation of adherens junctions. 3

17 and its loss is implicated in the epithelial mesenchymal transitions (EMT) that play a critical role during both normal embryonic development and tumor progression. Deletion of E cadherin in murine embryonic stem cells inhibits cell aggregation and induces lethality at the blastocyst stage (Larue et al., 1996). Homozygous mutant E cadherin embryos fail to form epithelial trophectoderm, which is the first sign of tissue differentiation in embryonic development (Ohsugi et al., 1997). This phenotype was not rescued by expression of another cadherin family member, N cadherin, suggesting that specific cadherins specify tissue type during development (Larue et al., 1996). Conditional gene targeting of E cadherin in the developing mouse epidermis results in perinatal lethality due to lack of a functional epidermal water barrier and loss of tight junctions (Tunggal et al., 2005). Likewise, conditional genetic ablation of α catenin in the mouse skin shows that loss of α catenin results in impaired adherens junction formation in vivo (Vasioukhin et al., 2001). p120 ablation in the mouse embryonic salivary gland led to reduced levels of E cadherin, as well as severe defects in adhesion, cell polarity, and acinar development, which ultimately resulted in formation of intraepithelial neoplasia (Davis and Reynolds, 2006). In addition to directly affecting cellular morphogenesis and motility, intercellular adhesion can also affect cell proliferation and apoptosis, which may regulate tissue formation and remodeling during development (Vleminckx and Kemler, 1999). The roles 4

18 of a few cell adhesion molecules during mammalian development have been identified by targeted gene inactivation in select tissues. Targeted inactivation of E cadherin in the lactating murine mammary gland prevents terminal differentiation and induces pervasive apoptosis (Boussadia et al., 2002). Conditional inactivation of the E cadherin gene in thyroid follicular cells, resulted in impaired development of the thyroid gland, however no loss of intercellular adhesion was observed, possibly due to compensation from other cadherins (Cali et al., 2007). Catenins also affect functions other than adhesion as targeted inactivation of α catenin in the skin epithelium of mice results in hyperproliferation (Vasioukhin et al., 2001). Thus, adherens junction proteins are required for multiple cellular processes, including intercellular adhesion, proliferation, and survival Adherens junctions in cancer Rarely do patients with solid, epithelial derived tumors die from the primary tumor; rather, they die from a metastatic lesion. Just as epithelial tissues require adherens junctions to maintain intercellular adhesion, so do tumors. Loss of intercellular adhesion allows tumor cells to disseminate from the primary lesion, escape into the bloodstream, and begin to grow at distal sites, thus initiating a metastatic growth. Therefore, targeting the adherens junctions either by preventing dissolution in the 5

19 primary tumor or preventing adherens junction formation at the distal sites represents an attractive therapeutic intervention to prevent death from cancer. Well differentiated, minimally invasive tumors retain strong E cadherin expression, however undifferentiated, invasive cancers with reduced intercellular adhesion often show reduced expression of E cadherin (Hirohashi and Kanai, 2003). The physiological consequence of E cadherin loss is that cells undergo an EMT resulting in a change from uniform, compact organized cells to amorphous cells that spread out and show reduced contact with one another (Birchmeier et al., 1996). Reduced E cadherin expression has been linked to increased tumor grade and increased patient mortality (Cavallaro and Christofori, 2004). E cadherin loss in tumors occurs through several mechanisms including upregulation of transcription repressors, hypermethylation of the cadherin promoter, mutations in the cadherin gene, signaling from oncoproteins, proteolytic degradation by matrix metalloproteases (MMPs), and upregulation of dysadherin, which has been shown to downregulate E cadherin (Cavallaro and Christofori, 2004; Hirohashi and Kanai, 2003; Huber et al., 2005). The increased tyrosine phosphorylaton of adherens junction components resulting from enhanced signaling from oncoproteins, such as v Src, or increased growth factor receptor signaling will be discussed in more detail later. β catenin is also frequently mutated in cancers, and may play a role in enhanced proliferation through regulation of the Wnt pathway (Miyoshi 6

20 and Hennighausen, 2002). Reduced expression of E cadherin and α catenin have also been observed in feline mammary tumors (Takauji et al., 2007). Likewise, reduced levels of E cadherin and disruption of β catenin have been found in canine mammary tumors (Restucci et al., 2007). The multiple levels at which cadherin mediated adhesion is regulated and the frequency with which adhesion is disrupted in cancer demonstrate its importance for tumor progression. Cadherin switching during the course of tumor progression is a phenomenon that has received increased attention. In several epithelial cancers, including breast and prostate cancer, as well as melanoma, N cadherin expression has been found to be upregulated (Hazan et al., 2004). Increased expression of N cadherin has been shown to promote morphological changes suggestive of EMT as well as increasing motility and invasiveness of breast cancer cells (Cowin et al., 2005). In addition to promoting cell motility, the cadherin switch may program cancer cells to home to a new site where they can adhere in an N cadherin dependent manner (Hazan et al., 2004). In the case of breast cancer, this could explain the high incidence of metastases to bone and brain. In axon guidance, the different cadherin types are thought to specify targets, e.g. N cadherin expressing neurons are attracted to N cadherin, but repelled from E cadherin (Thiery, 2003). Because of its correlation to increased invasion and metastasis, N cadherin has 7

21 become an attractive therapeutic target and at least one peptide based inhibitor has entered clinical testing (Mariotti et al., 2007) Adherens junction components Cadherin family proteins The classical cadherins consist of a family of single pass trans membrane glycoproteins, which mediate calcium dependent homotypic interactions with cadherin molecules on adjacent cells (Takeichi, 1994; Takeichi, 1995). Classical cadherins participate in adherens junction complexes in multiple tissues, and are named after the tissues in which they are most abundant. The most commonly studied mammalian members include epithelial (E ), neuronal (N ), placental (P ), and vascular endothelial (VE ), however multiple cadherins may be expressed in the same tissue. For example, both N and VE cadherin are expressed in endothelial cells and may act in concert to modulate intercellular adhesion (Luo and Radice, 2005). Extracellular calcium is thought to bind to the multiple extracellular cadherin motif subdomains, and stimulates cis dimerization or cadherin clustering (Steinberg and McNutt, 1999). Highly conserved HAV regions at the outermost cadherin motif (C1) are responsible for the transdimerization between cadherin molecules on neighboring cells (Koch et al., 1999; Pertz et al., 1999). The strengthening of intercellular adhesions relies on both cis and trans 8

22 interactions. Transcriptional control of E cadherin occurs through the binding of transcriptional factors to the E box of the E cadherin promoter (Perez Moreno et al., 2003). The transcription factors Snail and Slug bind to the E box to repress cadherin promoter activation, are critical for tissue morphogenesis during development, and promote EMT during tumor progression and metastasis (Perez Moreno et al., 2003). Regulation of cadherin at the protein level occurs through several mechanisms, which will be described in more detail below Catenin family proteins The mammalian catenin family of proteins was initially identified nearly 20 years ago and discovered in a complex with a cadherin family member (Ozawa et al., 1989). Catenins were thought to provide a link from the cadherin cytoplasmic tail to the actin cytoskeleton, and their name comes from the Latin catena for chain (Ozawa et al., 1989). The family includes of β-catenin and γ catenin (plakoglobin), p120 catenin and δ catenin, and α catenin. All catenin family members, with the exception of α catenin contain armadillo repeats, so named for their homology to the Drosophila β catenin like armadillo protein. β catenin and plakoglobin bind directly to the catenin binding domain, a 30 amino acid sequence at the cytoplasmic tail of cadherin molecules and as such are integral chains in the link to the cytoskeleton (Brunton et al., 2004). In addition to its role 9

23 in intercellular adhesion, β catenin participates in a number of signaling cascades, most notably Wnt signaling, and is found in complexes in the cytoplasm and nucleus (Perez Moreno and Fuchs, 2006). α catenin is linked indirectly to the cadherin complex by binding β catenin. α catenin provides the link to the actin cytoskeleton and was initially assumed to do so through direct binding to actin or through intermediate actin binding proteins like α actinin, formins, vinculin and others (Kobielak and Fuchs, 2004). While α catenin may regulate the actin cytoskeleton by binding to intermediates, it may also directly regulate actin filament organization by competing with the Arp2/3 complex for binding to actin filaments, thereby suppressing Arp2/3 mediated actin polymerization (Drees et al., 2005). p120 catenin binds directly to the juxtamembrane region of cadherin, and was initially identified as a protein tyrosine phosphorylated by Src kinase, and later identified as a member of the catenin family (Brunton et al., 2004). Subsequently, p120 catenin has been identified as a master regulator of adherens junctions through modulating the trafficking of cadherins (Reynolds, 2007). In addition to interacting with the cadherin catenin adhesion complex, p120 catenin has been shown to bind to or affect signaling downstream of numerous regulators of the actin cytoskeleton (Anastasiadis, 2007). While the catenins are required for intercellular adhesion through their participation in the cadherin catenin adhesion complex, there is clearly a need for 10

24 further study of the indirect role these proteins may play through other signaling cascades, which impinge on adherens junction regulation Actin cytoskeleton The actin cytoskeleton consists of many actin filaments of various lengths and acts to modulate the shape and motility of cells. It is highly dynamic and polymerization of actin monomers into filaments, followed by their cross linking into a 3 D meshwork is stimulated by a diverse set of physiological stimuli including stimulation by growth factors and adhesion molecules. Several protein families have been identified which directly modulate the polymerization and cross linking of actin, includingcomponents of the Arp 2/3 complex and formin family proteins. Cytoskeletal reorganization plays a critical role at each stage of adherens junction formation. Both classes of actin nucleator proteins, the Arp2/3 complex and the formins participate in de novo actin polymerization at various stages of adherens junction formation (Kobielak et al., 2004; Kovacs et al., 2002; Verma et al., 2004). Early formation of junctions is driven by the production of branched actin networks responsible for generating lamellipodial protrusions as a result of actin nucleation through Arp2/3 (Kobielak and Fuchs, 2004; Welch and Mullins, 2002). In contrast the formin family proteins nucleate unbranched actin filaments and are involved in the formation of the linear actin cables that radiate from early cadherin/catenin containing puncta (Kobielak 11

25 et al., 2004; Wallar and Alberts, 2003). A recent report suggests that the mammalian diaphanous related formin Dia1, a target of RhoA, is required for the reinforcement of adherens junctions (Carramusa et al., 2007). Actin polymerization provides the mechanical force which drives the formation of adherens junctions (Vasioukhin et al., 2000). The initial assembly of cadherindependent intercellular adhesions is stimulated by lamellipodial or filopodial membrane protrusions in adjacent cells (Ehrlich et al., 2002; Gavard et al., 2003; Kovacs et al., 2002; Vasioukhin et al., 2000). The engagement of cadherin molecules on neighboring cells, anchored to tips of lamellipodia and/or filopodia through the adhesion complex results in clusters of puncta or spot adherens junctions containing cadherins/catenins and other cytoskeletal proteins at sites of cell cell contact. Actin polymerization is stimulated at these early adherens junctions resulting in the formation of linear, radial actin cables which tether the adhesion complex at early junctions to the underlying cortical actin ring. The rate limiting step in the early stage of junction formation is, in fact, tethering the cadherin/catenin complex to the cortical actin cytoskeleton (Kobielak and Fuchs, 2004). Formation of these protrusive membrane structures requires the activity of Rho family GTPases, Rac and Cdc42, (described in more detail later) which stimulate formation of lamellipodia and filopodia, respectively (Etienne Manneville and Hall, 2002). Lamellipodia which form at puncta where cadherin molecules are engaged at 12

26 nascent cell adhesions are more persistent than at non contacting sites and also act to reinforce cadherin clustering (Ehrlich et al., 2002). The persistence of lamellipodia provides the force to close the adhesion zipper, resulting in a continuous line of cadherin/catenin complexes that is accompanied by reorganization of the actin cytoskeleton from a perpendicular to a lateral orientation parallel to the sealed membranes (Brunton et al., 2004). In addition to regulating adherens junction formation, Arp 2/3 dependent actin polymerization is also required for junction disassembly (Ivanov et al., 2005; Vaezi et al., 2002; Vasioukhin et al., 2000). In this regard, actin polymerization was shown to be required for dissolution of junctions in response to removal of extracellular calcium (Ivanov et al., 2005). The actin cytoskeleton also acts in concert with and regulates acto myosin contractility, microtubules and cell tension during adherens junction formation and dissolution (Mege et al., 2006) Regulation of adherens junctions The formation and maintenance of adherens junction require localization of members of the cell adhesion complex to the plasma membrane and binding of the cadherins/catenins to one another. While there are many unknown factors regarding the mechanisms through which localization and junctional integrity are regulated, several levels of regulation have been revealed. The members of the cadherin catenin complex regulate the localization of their binding partners; tyrosine phosphorylation of adherens 13

27 junction components regulates their localization and interactions; and the family of Rho GTPases play a critical role in regulating intercellular adhesion Adherens junction component binding The individual members of the adherens junction protein complex all play a role in regulating the localization of the others. Cadherin localization to the plasma membrane is required for localization of the catenins, and cadherin mutants lacking the β catenin binding site fail to promote intercellular adhesion (Nagafuchi and Takeichi, 1988). β catenin binding to cadherins is required for adherens junction formation to establish the link to α catenin. In this regard, the requirement for β catenin can be overcome by expression of a chimeric cadherin molecule fused to α catenin (Imamura et al., 1999; Ozawa, 1998). α catenin was traditionally believed to support intercellular adhesion by providing the link from the cadherin catenin complex either directly to the actin cytoskeleton or through intermediate actin binding proteins such as α actinin, vinculin, and ZO 1 (Bershadsky, 2004). However, this view has been challenged by the discovery that α catenin fails to bind actin while participating in a cadherin catenin complex (Drees et al., 2005). p120 catenin is essential for localization of cadherins and catenins to sites of cellcell contact and acts to regulate cadherin trafficking (Xiao et al., 2007). Following stimulation of adherens junction assembly, p120 catenin associates with kinesin and 14

28 essentially carries E cadherin to the plasma membrane along microtubules (Chen et al., 2003). In addition to facilitating adherens junction formation, p120 catenin is required for junctional stability. In all cell types examined, knockdown of p120 catenin led to decreased levels of cadherins, including E, N, P, and VE cadherin and resulted in loss of intercellular adhesion (Davis et al., 2003). In endothelial cells, p120 catenin binding to the cytoplasmic tail of VE cadherin is sufficient to prevent internalization by clathrinmediated endocytosis (Xiao et al., 2005). Conversely, selective uncoupling of p120 catenin from E cadherin increased the level of clathrin mediated endocytosis (Miyashita and Ozawa, 2007). However, E cadherin expression in cadherin deficient cells is sufficient to target p120 catenin to sites of cell cell contact, suggesting that the localization of E cadherin and p120 catenin to the plasma membrane is interdependent (Thoreson et al., 2000). Therefore, altered regulation of any individual member of the cadherin catenin complex may affect the others in various cell types and tissues Phosphorylation Tyrosine phosphorylation of adherens junction components plays a critical role in the regulation of junction formation and stability. A wealth of data suggest that phosphorylation of catenins downstream of activation of growth factors promotes the dissolution of adherens junctions, while interactions with various protein tyrosine phosphatases (PTPs) promote intercellular adhesion (Brunton et al., 2004; Steinberg and 15

29 McNutt, 1999). It is unknown whether catenins are direct substrates of tyrosine kinase growth factor receptors or are phosphorylated by downstream activated nonreceptor tyrosine kinases. Src is activated downstream of several growth factors, and cells transformed with v Src exhibit elevated levels of β catenin tyrosine phosphorylation, for example (Behrens et al., 1993; Esser et al., 1998). Tyrosine phosphorylation may not necessarily be causatively linked to disruption of adherens junctions. For example, lowlevel Src tyrosine kinase activity is required for the formation of adherens junctions in keratinocytes (Calautti et al., 1998). Likewise, tyrosine phosphorylation at sites of cellcell contact is transiently increased during adherens junction formation in endothelial cells, but is relatively low in confluent cells with mature junctions (Lampugnani et al., 1997). Cadherins (E, N, and VE ), β catenin, and p120 catenin are all targets of tyrosine phosphorylation (Brunton et al., 2004; Esser et al., 1998; Fujita et al., 2002; Siu et al., 2007). Expression of v Src promotes the tyrosine phosphorylation of E cadherin leading to decreased cell adhesion (Fujita et al., 2002). When phosphorylated, E cadherin recruits Hakai, an E3 ubiquitin ligase similar to Cbl, which targets E cadherin for ubiquitination and endocytosis (Fujita et al., 2002). Tyrosine phosphorylation of VEcadherin appears to promote maintenance of a mesenchymal cell morphology by 16

30 inhibiting binding of β catenin and p120 catenin (Potter et al., 2005). The functional consequence of phosphorylation of other cadherins remains an area of exploration. β catenin contains several tyrosine residues, which are known to be phosphorylated and lead to impaired intercellular adhesion. Abl mediated phosphorylation of tyrosine 489 has been proposed to disrupt binding to N cadherin (Rhee et al., 2007). Tyrosine phosphorylation of β catenin on residues 654 and 142 disrupts its association with E cadherin and α catenin, respectively (Brembeck et al., 2004; Piedra et al., 2003; Roura et al., 1999). Src kinases have been shown to directly phosphorylate tyrosine 654 however they do so inefficiently, suggesting that another kinase downstream of Src family kinases may phosphorylate this site (Piedra et al., 2003). In this regard, Fyn deficient keratinocytes exhibit decreased tyrosine phosphorylation of both β catenin and p120 catenin (Calautti et al., 1998). p120 catenin may function as a docking protein to recruit Fer, Fyn and Yes tyrosine kinases to the cadherin complex, which results in the phosphorylation of tyrosine 142 on β catenin (Piedra et al., 2003). Subsequently, an as yet unidentified tyrosine kinase (possibly Src, Fyn, or Abl) would then be recruited to the complex and phosphorylate β catenin on tyrosine 654, thereby disrupting its association with E cadherin (Piedra et al., 2003). Fer also serves to promote adhesion by phosphorylating PTP1B, which dephosphorylates tyrosine 654 (Xu et al., 2004). 17

31 The relative importance of tyrosine phosphorylation of p120 catenin is unclear. Tyrosine phosphorylation of p120 catenin is thought to be dispensable for its role in promoting adherens junction formation, however a recent report suggests that E cadherin targeted to the plasma membrane induces serine/threonine phosphorylation of p120 catenin (Fukumoto et al., 2007; Mariner et al., 2004). In addition to its direct interaction with the cadherin catenin complex, p120 catenin may regulate intercellular adhesion through its interaction with RhoA. In this regard, phosphorylation at tyrosine 112 by Fyn was shown to inhibit association of p120 catenin with Rho, whereas phosphorylation at tyrosines 217 and 228 by Src promoted the p120 catenin Rho interaction (Castano et al., 2007). Future studies will undoubtedly reveal new insights into the positive and negative roles played by the tyrosine phosphorylation of adherens junction components Rho GTPases Members of the family of RhoGTPases play a critical role in regulating the actin cytoskeleton, gene transcription, cell cycle progression, and membrane trafficking, and deregulation of RhoGTPase activity may contribute to tumorigenesis (Hall, 1998; Jaffe and Hall, 2002). The most well characterized family members are the small GTP binding proteins Rac1, Cdc42, and RhoA, although over 20 mammalian members have been identified. The phenotype of overexpression of activated forms of the Rho GTPases in 18

32 fibroblasts describes a general rule for what these proteins do: Rac1 mediates lamellipodia formation and membrane ruffling, Cdc42 mediates filopodia formation, and RhoA mediates stress fiber formation (Hall, 1998). RhoGTPases act indirectly through intermediate regulators, including Pak (downstream of Rac and Cdc42) and Rho activated kinase (ROCK, downstream of Rho) to effect cytoskeletal changes. Rho GTPase activity is stimulated by extracellular signals transduced through different classes of surface receptors including receptor tyrosine kinases, G proteincoupled receptors, cytokine receptors, and adhesion molecules (Kjoller and Hall, 1999). Rho proteins are in an active conformation when bound to GTP and inactive when bound to GTP. Rho guanine nucleotide exchange factors (Rho GEFs) promote the activation of Rho GTPases by catalyzing the exchange of GDP for GTP. Over 60 mammalian GEF family members have been described, with varying degrees of substrate selectivity. In addition to stimulating Rho activity, GEFs act to integrate signals, serve as signaling scaffolds, and can regulate intermolecular actions between Rho GTPases and other signaling cascades, e.g. heterotrimeric G proteins (Rossman et al., 2005). Rho GTPase activating proteins (Rho GAPs) function to activate the intrinsic GTPase activity of the Rho proteins, thus promoting turnover to the inactive, GDPbound state of the RhoGTPases. To date, over 70 Rho GAP family proteins have been 19

33 identified and cellular roles have been described in cell migration, cytokinesis, angiogenesis, differentiation, endocytosis, and more; however few family members have been studied in great detail (Tcherkezian and Lamarche Vane, 2007). p190 RhoGAP is a large GAP that acts solely on RhoA. Its activity is regulated by both protein protein interactions (binding to p120 Ras GAP) and tyrosine phosphorylation (Src/Arg) (Bradley et al., 2006; Bryant et al., 1995; Chang et al., 1995). p190 RhoGAP is known to regulate cytoskeletal changes induced by signals including PDGF and semaphorins, receptor signaling, integrin based adhesion, and intercellular adhesion (Barberis et al., 2005; Bradley et al., 2006; Noren et al., 2003; Wildenberg et al., 2006). Rho guanine nucleotide dissociation inhibitors (Rho GDIs) act at a different level and regulate the cytosol/membrane bound state of Rho GTPases. RhoGDIs physically interact with Rho GTPases to maintain a pool of inactive Rho GDP in the cytoplasm of resting cells by preventing the spontaneous dissociation of GDP from the GTPases (Olofsson, 1999). In contrast to GEFs and GAPs, the Rho GDI family consists of only 3 family members. However, it is possible that other proteins which bind to Rho family members could perform the same function. The biological function of GDIs remain under studied, however GDIs have recently been implicated in tumor migration and invasion as well as in bacterial infection of Clostridium difficile (Dransart et al., 2005). The 20

34 dearth of knowledge combined with the abundance of GEFs, GAPs, and GDIs denotes a long road ahead for those studying the complex regulation of RhoGTPAses. Rac1, Cdc42, and RhoA have been implicated in regulating the formation, maintenance and turnover of cadherin based cell cell adhesion (Fukata and Kaibuchi, 2001). Rac has been shown to both positively and negatively regulate adherens junctions in various contexts depending on cell type, level of activated Rac, maturity of the adherens junction, and the type of cadherin (Lozano et al., 2003). Rac is rapidly activated within minutes following stimulation of cadherin engagement and is transiently recruited to nascent adherens junctions (Ehrlich et al., 2002). Both Rac and Cdc42 regulate E cadherin trafficking to the membrane in MDCK cells, as expression of dominant negative Rac and Cdc42 results in E cadherin accumulation at a distinct post Golgi step prior to its association with p120 catenin (Wang et al., 2005). Rac and Cdc42 also act to retain E cadherin at sites of cell cell contact by blocking its endocytosis (Izumi et al., 2004). Conversely, sustained activation of Rac in keratinocytes was sufficient to disrupt adherens junctions and resulted in re localization of cadherin molecules from the plasma membrane (Braga et al., 2000). Therefore, the role of Rac in modulating adherens junctions likely depends on the complex integration of multiple cellular signals. 21

35 Similarly, RhoA and its effector kinase ROCK, have been shown to to both positively and negatively regulate adherens junctions (Lozano et al., 2003; Sahai and Marshall, 2002). Confluent monolayers of epithelial cells show decreased levels of Rho activity, and Rho activity is inhibited following the stimulation of cadherin engagement (Noren et al., 2001). Cadherin engagement leads to enhanced tyrosine phosphorylation of p190 RhoGAP, which likely stimulates its GAP activity (Noren et al., 2003). In this regard, PP2 treatment, which pharmacologically inhibits the activity of both Src and Abl kinases (Tatton et al., 2003), abolishes the binding of p190 RhoGAP to activated RhoA, (Noren et al., 2003). Further, inhibition of RhoA activity by cadherin engagement is blocked by expression of a dominant negative p190 RhoGAP (GAP deficient) (Noren et al., 2003). p190 RhoGAP may act to regulate adherens junctions through interactions with other molecules. Rac induced stimulation of adherens junctions in fibroblasts was recently shown to result in binding of p190 RhoGAP to p120 catenin and their colocalization at sites of cell cell contact (Wildenberg et al., 2006). Rac induced adherens junctions failed to form in fibroblasts lacking p190 RhoGAP (Wildenberg et al., 2006). Similarly, RhoA has been reported to positively regulate adherens junction formation and stability in keratinocytes (Perez Moreno et al., 2003). In contrast, increased Rho activity may lead to increased cell contractility, thereby increasing tensile stress at cell cell junctions, ultimately resulting in their 22

36 destabilization (Paszek et al., 2005). The functional consequence of activating RhoA and its ability to both positively and negatively regulate cell cell adhesion may be explained by context dependent activation of different downstream effectors. Activation of the Rho effector diaphanous related formin Dia1 is required for the stability of adherens junctions (Sahai and Marshall, 2002). However, activation of the ROCK kinase downstream of active Rho results in acto myosin contractility and disruption of adherens junctions (Sahai and Marshall, 2002). The consequences of Rho activation on regulation of cell cell adhesion may only be understood in the context of integrating all of the other cellular signals feeding into the Rho pathway. Gaining a better understanding of how individual Rho GEFs, GAPs, and GDIs regulate Rac, Cdc42, and Rho control of intercellular adhesion may allow these apparent paradoxes to be resolved. 1.2 Cross-talk between Adherens junctions and Receptor Tyrosine Kinases Receptor tyrosine kinases (RTKs) are a special class of growth factor receptors and have been shown to play a critical role in various cellular processes including cell proliferation, survival, motility, and directed migration (Schlessinger, 2000). A role for RTKs in the modulation of adhesion was first discovered in the cross talk between growth factor receptors and integrin adhesion molecules (Brunton et al., 2004). More 23

37 recent evidence points to a critical role for RTKs in modulating changes in intercellular adhesion through interactions with the cadherin based adherens junctions (Brunton et al., 2004). Cross talk between RTKs and adherens junctions is particularly critical during tumor progression, therefore understanding this phenomenon may lead to the discovery of novel therapeutic targets (Cavallaro and Christofori, 2004; Hazan et al., 2004). The family of RTKs includes receptors which are commonly named for the specific growth factor ligands to which they respond: epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR). All RTKs possess a kinase domain, which endows intrinsic protein tyrosine kinase activity, and a number of tyrosine residues which may be phosphorylated and contribute to regulation (Schlessinger, 2000). Individual RTKs are expressed preferentially in different cell types and may act differently when stimulated in one cell type vs. another. However, they share a common signaling mechanism and often activate a common set of downstream signaling pathways. RTKs are activated by ligand induced receptor dimerization leading to both auto and transphorylation of the receptor and resulting in enhanced tyrosine kinase activity (Schlessinger, 2000). Phosphorylated tyrosines on the receptor cytoplasmic tail serve as docking sites for the recruitment of additional signaling molecules containing 24

38 SH2 (Src homology 2) or PTB (phosphotyrosine binding) domains, and these molecules are frequently substrates of the receptor (Schlessinger, 2000). Common signaling modules activated downstream of growth factor receptor activation include the Ras/MAP Kinase signaling cascade, phosphoinositol metabolism and downstream signaling through the PI 3 kinase pathway, and gene transcription through nuclear translocation of STATs (Schlessinger, 2000). In addition, nonreceptor tyrosine kinases (NRTKs) which are recruited to activated receptors may phosphorylate the RTK in trans to alter signaling. In this regard, both Src and Abl are activated by EGFR activation, and activated Src and Abl have been shown to phosphorylate EGFR (Tanos and Pendergast, 2006). Numerous studies suggest the aberrant cell signaling induced by hyperactivation of RTKs and their downstream targets promotes tumor progression of many epithelial cancers through effects on cell proliferation, survival, and motility (Christensen et al., 2005; Normanno et al., 2006; Shinkaruk et al., 2003). RTKs also promote tumor progression by regulating cadherin based adhesion (Brunton et al., 2004; Cavallaro and Christofori, 2004) Interactions between adherens junctions and RTKs Reciprocal signaling between adherens junctions and RTKs remains poorly understood; however, three themes of cross talk have emerged. RTK signaling is affected by the presence and strength of intercellular adhesion; RTKs regulate the 25

39 localization of adherens junctions components; and RTKs modulate the tyrosine phosphorylation of adherens junctions components (Comoglio et al., 2003). In a limited number of cases, cadherins have been shown to regulate signaling by growth factor receptors. Stimulation of FGFR1 with FGF results in the cadherindependent co endocytosis of E cadherin and FGFR1, followed by nuclear translocation of FGFR1 (Bryant and Stow, 2004). The effect of FGF is overridden with overexpression of E cadherin or p120 catenin, which stabilizes protein levels of E cadherin (Bryant and Stow, 2004). Conversely, stimulation of cadherin based adhesion can lead to association of EGFR with E cadherin, which promotes the autophosphorylation of EGFR (Pece and Gutkind, 2000). In this regard, cadherin engagement is required for signaling from the EGFR through stimulation of both the MAPK cascade and Rac activation (Betson et al., 2002; Pece and Gutkind, 2000). The effects of cadherin engagement on EGFR appear to be dependent on cell density as activation of EGFR and EGFR dependent proliferation are inhibited in cells with mature adherens junctions (Curto et al., 2007). However, cells lacking NF2, which mediates cross talk between the EGFR and adherens junctions, display persistent EGFR activation and fail to undergo growth arrest at high cell density (Curto et al., 2007). This paradox whereby cadherin both promotes and arrests EGFRmediated cell proliferation is a common theme in the cross talk between RTKs and 26

40 adherens junctions and points to the complexity of understanding interactions between different signaling molecules. There are numerous examples in which RTKs have been shown to regulate adherens junctions through direct physical interactions with or indirect regulation of cadherin molecules. For example, EGF stimulation of the EGFR has been shown to increase endocytosis of E cadherin through caveolin, and chronic EGF stimulation can decrease E cadherin expression, thereby increasing the invasive potential of tumor cells (Lu et al., 2003). Likewise, both FGF1 and FGF2 stimulation result in E cadherin internalization and re localization of β catenin from the cell membrane to the cytoplasm and nucleus in NBT II cells (Billottet et al., 2004). Loss of E cadherin based cell cell contacts in epithelial cells over the course of tumorigenesis leads to both morphological changes and increased invasive and metastatic potential of the cells (Cavallaro and Christofori, 2004). Cadherin switching is a well documented process in various tumors of epithelial origin where mesenchymal cadherins, especially N cadherin, are spontaneously expressed following the loss of E cadherin (Bonitsis et al., 2006; Cavallaro et al., 2002; Hazan et al., 2004). Cancer cells expressing N cadherin tend to be more invasive, but the molecular mechanisms underlying this switch and its effects have yet to be defined. Evidence from some epithelial cell lines suggests that N cadherin physically interacts 27

41 with FGFR to enhance FGF2 stimulated signaling and invasion (Suyama et al., 2002). Additionally, a soluble fragment of N cadherin has been shown to promote angiogenesis in an N cadherin and FGFR dependent manner in endothelial cells (Derycke et al., 2006). In neuronal cells, N cadherin has been shown to affect axon guidance through the formation of an intermolecular complex with the Robo receptor, mediated by Abl (Rhee et al., 2007; Rhee et al., 2002). Recently, Robo and its ligand Slit have been shown to be upregulated in some breast and prostate cancers (Dallol et al., 2002; Latil et al., 2003). In the brain, Slit mediates repulsion, whereas in breast cancer, it has been shown to promote chemotaxis (McAllister, 2002; Prasad et al., 2004). The interplay between FGFR and Robo with N cadherin warrants further study as both FGF and Slit have been implicated in tumor angiogenesis (Fujiwara et al., 2006; Presta et al., 2005). Activation of growth factor receptors can lead to tyrosine phosphorylation of adherens junctions components. In endothelial cells, tyrosine phosphorylation of adherens junction components, including VE cadherin, β catenin and p120 catenin, is dynamically regulated during the formation and maturation of cell cell contacts (Esser et al., 1998; Lampugnani et al., 1997). VEGF stimulation leads to enhanced tyrosine phosphorylation at endothelial cell cell contacts concomitant with increased tyrosine phosphorylation of adherens junction components, including VE cadherin, β catenin, and p120 catenin but not α catenin (Esser et al., 1998). This phosphorylation may be 28

42 important for VEGF induced cell permeability by promoting the dissolution of cell cell contacts. Similarly, activation of the Roundabout (Robo) receptor following binding of its ligand Slit modulates adhesion by promoting tyrosine phosphorylation of β catenin resulting in dissociation of β catenin from N cadherin (Rhee et al., 2002). In this context, the enhanced phosphorylation of β catenin on tyrosine 489 has been reported to be dependent on Abl kinase (Rhee et al., 2007). Tyrosine phosphorylation of β catenin is enhanced following growth factor stimulation with EGF and IGF 1, and p120 catenin is phosphorylated in response to EGF and PDGF (Brunton et al., 2004). It is unclear whether cadherins and catenins are direct substrates of the RTKs or whether they are phosphorylated through other tyrosine kinases, such as Src or Abl Met receptor tyrosine kinase Cancer relevance Dynamic remodeling of cell cell adhesions plays a critical role in normal development and is also required for tumor invasion and metastasis (Thiery, 2002). Hepatocyte growth factor (HGF), also known as scatter factor (SF), was first characterized as a physiological factor promoting cell scattering through the remodeling of adherens junctions (Birchmeier et al., 2003; Stoker et al., 1987). Subsequently, HGF has been identified as a critical mediator of tumor progression in various epithelial derived 29

43 tumors (Hurle et al., 2005; Jiang et al., 2005) and has been implicated in tumor angiogenesis (Rosen et al., 1997). The receptor for HGF, Met, is upregulated in human breast and prostate cancer patients and is known to promote metastasis (Birchmeier et al., 2003; Knudsen and Edlund, 2004). Met is also upregulated in canine prostate cancer, and the Met family member Stk, a homologue of RON, is upregulated in feline breast carcinoma (De Maria et al., 2002; Liao et al., 2005). Met may also play a role in tumor progression through upregulation of its activity. Recently, a somatic mutation was identified in lung cancer patients, which leads to prolonged Met signaling (Kong Beltran et al., 2006). HGF Met signaling has received increasing attention for its role in tumor angiogenesis suggesting that Met may play a role in cancers in which it has not yet been implicated (Lesko and Majka, 2008). The fact that increased Met activity and upregulation of Met and its family members in spontaneous cancers in various species have been observed underscores its importance as a potential therapeutic target Biological effects of Met activation Acquisition of an invasive phenotype is a critical step in metastatic progression. HGF acts at several levels to promote epithelial mesenchymal transition (EMT) by stimulating breakdown of cell cell junctions and increasing cell motility and invasiveness (Thiery, 2002). As the first phenotype attributed to HGF stimulation, cell scattering is fairly well understood, however there are likely to be unidentified 30

44 components to the pathway, particularly linking Met activation to regulation of the family of RhoGTPAses. Cell scattering in MDCK cells occurs in a step wise manner following HGF stimulation. Within minutes after the Met receptor is activated, cortical actin begins to break down and cells form lamellipodia and spread. At this point, redistribution of β catenin to the cytoplasm from sites of cell cell contact is initiated and the cells begin to detach from one another. On the order of hours following Met stimulation, cells form actin stress fibers and begin to acquire a mesenchymal morphology and become more motile. In addition to its well characterized role in cell motility, migration, and invasion, HGF stimulation of the Met receptor also leads to increased cell proliferation. However, a recent report suggests that the downstream signals elicited by Met activation may differentiate between the two phenotypes: proliferation is regulated by activation of the c myc pathway, whereas invasion is mediated through activation of the MAPK pathway (Gao et al., 2005). In a more physiologically relevant context, HGF can induce the formation of branched tubules when epithelial cells are cultured within a three dimensional collagen matrix in vitro (Rosario and Birchmeier, 2003; Stella and Comoglio, 1999). Initially, individual cells seeded in a collagen matrix proliferate and form hollowed out spherical cysts. When these cysts are stimulated with HGF, they form tubules in a step wise manner (Pollack et al., 1997). First, individual cells undergo partial EMT and extend cell 31

45 processes. This is followed by the extension of cell chains from the cyst. The cells in the chain then elongate and form cords and begin the process of redifferentiation in which the cells reorganize into a hollowed out tube, projecting out of the cyst. In this regard, prolonged stimulation with HGF leads to the step wise activation of signaling cascades, which are critical at different stages of the process (OʹBrien et al., 2004). During the initial phases of tube sprouting, cells undergo a partial EMT which is dependent on Erk activation; during redifferentiation, MMP activation is required, however Erk activity is maintained but is not required for this process (OʹBrien et al., 2004). An in vivo role for HGF was demonstrated in tubule formation during liver and kidney regeneration, and mammary gland formation (Rosario and Birchmeier, 2003) Met signaling A critical distinction between Met and other growth factor receptors is the distinct program of invasive growth elicited downstream of its stimulation. This unique activity may be due to the prolonged activation of signaling molecules such as Erk by HGF compared to the more transient activation induced by growth factors such as EGF (Comoglio and Trusolino, 2002). Additionally, the diversity in number and combination of signaling molecules recruited directly to the Met receptor or indirectly through other proteins may allow HGF stimulation to produce distinct biological responses in different cellular contexts. 32

46 HGF binding to the Met receptor tyrosine kinase induces dimerization and autophosphorylation of the receptor. Phosphorylation of tyrosine residues 1234 and 1235 in the catalytic domain are critical for Met activation (Longati et al., 1994; Rodrigues and Park, 1994). Other phosphorylated tyrosine residues, including tyrosines 1349 and 1356, serve as docking sites for multiple SH2 domain containing signaling molecules which are recruited to the Met receptor (Ponzetto et al., 1994). Signaling molecules recruited to Met include Grb2, PI3 kinase, Src, PLC γ, Cbl, Crk, and Gab1. Gab1 is a large adaptor molecule which is tyrosine phosphorylated and recruited to the Met receptor following Met activation (Weidner et al., 1996). Gab1 likewise contains a number of tyrosine residues which, when phosphorylated, serve as docking sites for the recruitment of additional signaling molecules and is required for Met signaling (Bardelli et al., 1997; Sakkab et al., 2000). Signal attenuation is less well understood but is mediated in part through Cblmediated downregulation of the Met receptor (Petrelli et al., 2002). Ubiquitination and internalization of Met requires Cbl binding through its tyrosine kinase binding domain to phosphorylated tyrosine 1003 of the Met receptor (Peschard et al., 2004). A ubiquitindeficient Met receptor mutant in which this tyrosine has been mutated to phenylalanine (Met Y1003F) shows increased receptor stability, prolonged activation of signaling pathways, and enhanced transformation in response to HGF stimulation (Abella et al., 33

47 2005). The finding that mutations in the Cbl binding region of Met have been observed in multiple tumors supports a critical need for understanding the role of phosphorylation of Met Y1003 (Peschard and Park, 2003). Crk is one of several SH2 domain containing adaptor molecules recruited to the Met receptor by binding Gab1. Both Crk and its family member CrkL have been shown to bind tyrosine phosphorylated YXXP motifs on Gab1 (Lamorte et al., 2002b; Sakkab et al., 2000). Crk is required for the morphological changes induced in epithelial cells in response to HGF treatment, and overexpression of Crk in MDCK cells promotes cell spreading, induction of lamellipodial protrusions, activation of Rac, and dissolution of adherens junctions, thus mimicking HGF mediated effects (Lamorte et al., 2002b). Tyrosine phosphorylation of Crk increases in response to HGF treatment, and this phosphorylation correlates with increased binding of Crk to p130cas, Cbl, and Gab1 (Lamorte et al., 2002b). Crk was shown to be required for the sustained phosphorylation of Gab1 at Y307 and sustained Met signaling in response to HGF treatment (Watanabe et al., 2006). More recent data suggest that Crk participates in a negative regulatory feedback loop with Met. In this regard, Met signaling induces Abl dependent tyrosine phosphorylation of Crk at Y221, and this phosphorylation is required to attenuate Met signaling in fibroblasts (Cipres et al., 2007). Thus, Crk appears to be required for activation of both Met signaling and signal attenuation. 34

48 RhoGTPases regulate cytoskeletal rearrangements downstream of signaling by numerous growth factors, including HGF. Both Rac and Cdc42 are activated and are required for cell spreading following HGF stimulation of MDCK cells (Ridley et al., 1995; Royal et al., 2000). Activation of Rac as well as the breakdown of junctions in MDCK cells following Met activation is inhibited by treatment with a PI 3 kinase inhibitor, suggesting a critical role for the PI 3 kinase pathway in some contexts (Royal et al., 2000; Royal and Park, 1995). Rac activation is enhanced in cells overexpressing Crk, and is required for Crk induced lamellipodia formation and cell spreading (Lamorte et al., 2002b; Royal et al., 2000). A recent study suggests that Met overexpression leads to Rac dependent generation of reactive oxygen species (ROS), and that treatment with ROS scavengers blocks signaling to Erk (Ferraro et al., 2006). Generation of ROS through Rac may occur through increased levels of of the Nox family of NAD(P)H oxidases (Paravicini and Touyz, 2008), which has also been shown to operate downstream of Abl kinase activation (Boureux et al., 2005). In addition, this novel Met ROS pathway is required for the in vivo pro metastatic behavior of Met (Ferraro et al., 2006). Less is known regarding a potential role for Rho signaling downstream of Met activation, however its downstream target ROCK has been implicated in one study (Royal et al., 2000). ROCK translocates to membrane ruffles in response to HGF stimulation in MDCK cells and is required for formation of 35

49 lamellipodia, stress fiber formation, and reorganization of focal adhesions (Royal et al., 2000). However, cell spreading occurs independently of ROCK (Royal et al., 2000). The question of how signals are transduced from Met to the RhoGTPases remains open. The mechanism through which Met activation results in adherens junction dissolution is unclear, however, it may be due in part to regulation of adherens junction components. HGF stimulation of Met results in increased tyrosine phosphorylation of β catenin (Hiscox and Jiang, 1999; Monga et al., 2002). Tyrosine phosphorylation of β catenin is known to alter its interaction with E cadherin and, in some cases, to disrupt the cadherin catenin complex (Lilien and Balsamo, 2005). Further, Met has been shown to interact physically with both β catenin and E cadherin (Birchmeier et al., 2003). Significantly, expression of E cadherin in cells lacking E cadherin stimulated relocalization of the Met receptor from intracellular compartments to the plasma membrane (Reshetnikova et al., 2007). A role for reciprocal signaling between the Met receptor and E cadherin, particularly during tumor progression, remains unexplored. 1.3 Abl family of non-receptor Tyrosine Kinases The founding member of the Abl protein family, Abl, was originally identified as the cellular homolog of the v Abl oncogene product of the Abelson murine leukemia virus (A MULV) (Goff et al., 1980; Wang et al., 1984). The Abl family of non receptor tyrosine kinases consists of two mammalian members c Abl and Arg, (Goff et al., 1980; 36

50 Heisterkamp et al., 1983; Kruh et al., 1990), Drosophila Abl (D Abl) (Hoffman et al., 1983), and C. elegans Abl (Goddard et al., 1986). Abl is perhaps best well known for its role in human leukemias as part of the Philadelphia chromosome resulting from chromosomal translocation of the two genes Bcr and Abl (Groffen et al., 1984; Melo, 1996). Both v Abl and BcrAbl are capable of inducing transformation in various cell types, and have been shown to affect cell proliferation and survival pathways, as well as cell adhesion and migration function (Pendergast, 2002). While there have been far fewer studies describing the normal functions of cellular Abl and Arg, a growing body of evidence suggests that normal functions also include regulation of cell proliferation, survival, response to extracellular stresses, cytoskeletal rearrangments, and adhesion in various tissues (Pendergast, 2002) Abl and Arg Abl and Arg show ~90% sequence identity in their SH3, SH2, and kinase domains suggesting that they likely share binding partners and substrates (Pendergast, 2002) (Figure 2). The SH3 domain recognizes proline rich sequences with the consensus PPXXXPPXXP, where P is proline and X any amino acid (Alexandropoulos et al., 1995; Cicchetti et al., 1992; Ren et al., 1993; Rickles et al., 1994), while the SH2 domain recognizes the YXXP sequence where Y is a phosphorylated tyrosine, X any amino acid, and P proline (Songyang et al., 1993). The kinase domain preferentially phosphorylates 37

51 peptides with the consensus sequence I/VYXXP, where I is isoleucine, V valine, Y tyrosine, X any amino acid, and P proline (Pendergast, 2002). Further highlighting the conserved evolution of substrate specificity, the kinase domain of Drosophila Abl is about 77% identical to that of mammalian Abl and Arg (Henkemeyer et al., 1988). Abl family proteins diverge in the C terminal regions. In spite of the diversity, there are several proline rich sequences downstream of the kinase domain which are highly conserved among Abl and Arg (Pendergast, 2002). Likewise, both Abl and Arg contain actin binding domains and bundle F actin in vitro which may play a role in modulating cytoskeletal rearrangements (Baum and Perrimon, 2001; Galkin et al., 2005; Wang et al., 2001; Woodring et al., 2002). More recently, Arg has been shown to bind microtubules and to play a role in lamellipodial dynamics through regulating crosslinking between actin bundles and microtubules (Miller et al., 2004). A key distinction between Abl and Arg is the presence of three nuclear localization sequences (NLSs) in Abl (Van Etten et al., 1989; Wen et al., 1996), which are not present in D Abl (Henkemeyer et al., 1988). Abl also contains a functional nuclear export sequence (NES) (Taagepera et al., 1998), which allows for nuclear cytoplasmic shuttling, as well as a DNA binding domain (David Cordonnier et al., 1998; Kipreos and Wang, 1992; Miao and Wang, 1996). This suggests that mammalian Abl has evolved and diverged from 38

52 Arg in response to a need for regulating some nuclear function not required in lower organisms. 39

53 NLS Binding sites for: c-abl b form V SH3 SH2 KINASE DNA G-actin F-actin a form NES SH3 Domain Binding Sites Proline Stretch Binding sites for: b form F-actin microtubules G-actin F-actin Arg V SH3 SH2 KINASE a form NES 32% 90% 94% 29% 56% Figure 2: Functional domains of c Abl and Arg. Adapted from Pendergast

54 Both Abl and Arg are expressed in a wide variety of fetal and adult tissues (Courtney et al., 2000; Gertler et al., 1993; Koleske et al., 1998; Muller et al., 1982; OʹNeill et al., 1997; Perego et al., 1991). Arg appears to be more highly enriched in the mammalian brain and CNS in flies as well as in muscle, thymus, and spleen (Gertler et al., 1993; Koleske et al., 1998). Abl appears to be preferentially expressed in cartilage, adipocytes, and ciliated epithelium in adult tissues (OʹNeill et al., 1997). Abl and Arg also exhibit similarities and differences in subcellular localization (Pendergast, 2002). Whereas Abl has been shown to localize to the plasma membrane, cytosol, endoplasmic reticulum, mitochondria, lipid rafts, actin cytoskeleton and the nucleus in response to different stimuli (Frasca et al., 2001; Ito et al., 2001; Koleske et al., 1998; Lewis et al., 1996; OʹNeill et al., 1997; Plattner et al., 1999; Van Etten et al., 1989; Westphal et al., 2000; Wetzler et al., 1993; Zipfel et al., 2000), Arg is primarily observed in the cytoplasm and cytoskeleton (Koleske et al., 1998; Wang and Kruh, 1996; Wang et al., 2001). More recently, both exogenous Abl and BcrAbl have been shown to localize to nascent adherens junctions when expressed in Drosophila (Stevens et al., 2007). The mechanism by which fractions of the total pool of cellular Abl and Arg are dynamically re localized to different compartments remains unclear, however localization studies often provide the first clues to discovering novel normal cellular functions for Abl and Arg. 41

55 1.3.2 Regulation of Abl/Arg The Abl kinases have been implicated in a wide variety of cellular processes as described above. Because the Abl tyrosine kinases can be found throughout the cell, and one or both are expressed in nearly every cell type, regulation of Abl kinase activity must be tightly restricted. This regulation occurs at several levels include physical intraand intermolecular interactions and protein modifications including phosphorylation and ubiquitination, which act to regulate both basal and stimulated Abl kinase activity Physical Activation of Abl kinases may occur through the binding of several proteins involved in various signal cascades (Pendergast, 2002). In the context of Ras activation, Rin1 was recently shown to bind Abl resulting in increased kinase activity (Hu et al., 2005). Overexpression of the adaptor proteins Nck and Crk in 293T cells has been shown to increase Abl kinase activity, and is dependent on the SH3 domains of both proteins, however the exact mechanism is unknown (Shishido et al., 2001; Smith et al., 1999). Overexpression of several other Abl interacting proteins, including Cbl, c Jun, and Abi family proteins, increases Abl activity through the relief of inhibitory interactions (Dai and Pendergast, 1995; Shi et al., 1995). Intra and intermolecular actions also play a key inhibitory role in regulating cellular Abl kinase activity. In part, Abl is held in the inhibited state by a physical block 42

56 of the kinase domain by the SH3 SH2 domain folding back over it. A recent analysis of Abl crystal structure in the autoinhibited state revealed that a previously unseen N terminal cap segment adjacent to the SH3 SH2 domains is required to maintain the link between SH3 SH2 and the kinase domain (Nagar et al., 2006). Both deletion and mutation of the Abl SH3 domain have been shown to stimulate Abl kinase activity in vivo and result in enhanced tyrosine phosphorylation of Abl (Jackson and Baltimore, 1989). While the relative importance or interdependence of each interaction is unknown, inhibitory interactions between the SH3 domain and the linker region connecting the SH2 are important for the negative regulation of Abl kinase activity (Barila and Superti Furga, 1998; Brasher et al., 2001). Additionally, binding of the Abl kinase domain to an NH2 terminal myristoyl residue provides an additional structural mechanism for negative regulation (Nagar et al., 2003). A number of proteins have been identified that negatively regulate Abl kinase activity through binding to various domains. Proteins that inhibit Abl kinase activity through binding to the SH3 domain include Pag/Msp23 and AAP1 (Wen and Van Etten, 1997; Zhu and Shore, 1996). The retinoblastoma protein, Rb, binds to the ATP binding lobe of the kinase domain of Abl in the nucleus (Welch and Wang, 1993). F actin has been shown to inhibit Abl kinase activity by binding to the extreme carboxy terminus of Abl (Woodring et al., 2002). The diversity of known physical methods of regulating Abl 43

57 activation and the possible existence of as yet unidentified methods highlight the critical importance of modulating cellular levels of Abl activity Protein modification In addition to physical regulation, protein modifications play a key role in regulating Abl kinase activity. Tyrosine phosphorylation of Abl robustly stimulates its catalytic activity. For example, Abl activity is increased 10 to 20 fold in cells expressing activated Src kinases (Plattner et al., 1999). The Src kinases directly phosphorylate Abl at tyrosine 412, and phosphorylation of this site is required for enhanced Abl kinase activity in the context of expression of activated Src Y527F (Plattner et al., 1999) Further, autophosphorylation of Abl results in an 18 fold increase in activity, which is reduced by 90% when tyrosine 412 is mutated to phenylalanine (Brasher and Van Etten, 2000). Mutation of a second tyrosine residue, tyrosine 245, also resulted in a 50% decreased activation of wild type Abl suggesting this residue may also be important. The crystal structure of the catalytic domain of c Abl in a complex with STI 571, which is a small molecule inhibitor of the Abl kinases, also suggests the critical importance of tyrosine 412 (Pendergast, 2002). In complex with STI 571, tyrosine 412 in the activation loop of Abl points inward decreasing accessibility to other kinases (Schindler et al., 2000). Negative regulation of Abl kinase activity following stimulation is critical to restrict the signal duration and location. Evidence in fibroblasts lacking the PEST type 44

58 phosphotyrosine phosphatases (PTPases) suggest they act to dephosphorylate Abl (Cong et al., 2000). Similarly, treatment of unstimulated cells with the protein tyrosine phosphatase inhibitor orthovanadate results in enhanced tyrosine phosphorylation of Abl suggesting that tyrosine phosphatases are responsible for maintaining relatively low levels of endogenous Abl kinase activity (Cong et al., 2000; Dorey et al., 2001; Echarri and Pendergast, 2001). Phosphorylation at tyrosines 245 and 412 also serves to regulate protein stability as mutation of these tyrosines to phenylalanine results in increased protein stability (Echarri and Pendergast, 2001). This effect is likely to be mediated through ubiquitin mediated degradation. Inhibition of the 26s proteasome leads to both enhanced tyrosine phosphorylation and enhanced protein stability of Abl (Echarri and Pendergast, 2001). Together, dephosphorylation and ubiquitin mediated degradation act to tightly control spatial temporal activation of stimulated and basal Abl kinase activity Upstream signals regulating Abl tyrosine kinase activity Abl and Arg are activated by a multitude of extracellular and intracellular signals and have been shown to participate in signaling cascades involving a number of cellular processes (Pendergast, 2002) (Figure 3). Analysis of Abl kinase activity during the progression of cells from quiescence to S phase revealed that the nuclear pool of Abl is activated during S phase suggesting a role for Abl in cell cycle regulation (Welch and Wang, 1993). Nuclear Abl is also activated in response to DNA damage (Kharbanda et 45

59 al., 1995; Liu et al., 1996) and may play a role in homologous recombination (Li et al., 2002). Oxidative stress is another potent activator of Abl kinase activity. Treatment of cells with hydrogen peroxide activates the cytoplasmic pool of Abl (Sun et al., 2000b; Sun et al., 2000c). This process may be modulated by S glutathionylation of cysteine residues on Abl induced by reactive oxygen species (Leonberg and Chai, 2007). The functional consequences of Abl activation in this context are not well understood, yet Abl may act in part by activating glutathione peroxidase 1 to protect cells against damage induced by oxidative stress (Cao et al., 2003). Generation of reactive oxygen species cancer by growth factor receptors, cytokines, and other stimuli, including Rac activation, has been proposed to promote metastasis (Finkel, 2006; Wu, 2006). A potential role for Abl kinases in this context remains untested. Extracellular signals transmitted through receptor tyrosine kinases (discussed in more detail below) and adhesion molecules also activate the Abl kinases (Lewis et al., 1996; Pendergast, 2002). The Abl kinases are required for a variety of integrin dependent cellular process including dendritic branching in response to laminin 1 or Semaphorin 7A (Moresco et al., 2005), mammary epithelial cell adhesion (Hu et al., 2005), and fibroblast spreading (Miller et al., 2004; Woodring et al., 2002; Woodring et al., 2004). Arg has been shown to regulate adhesion dependent tyrosine phosphorylation of p190rhogap in neuronal cells, which is critical for neuronal morphogenesis in the 46

60 Activated by: PDGF, EGF, NGF, Agrin TCR activation Bacterial Invasion Src family Oxidative stress DNA Damage Integrins c-abl c-abl PIP 2 Tyrosine Phosphatases P P E3 Ub Ub Ub Ub Deactivated by: Degradation by the 26S proteasome Figure 3: Regulation of Abl kinase activity. Adapted from Pendergast

61 developing mouse brain (Hernandez et al., 2004). Arg dependent phosphorylation of p190rhogap downstream of integrin engagement is required to inhibit Rho activity locally at the cell periphery in order to allow fibroblasts to initiate cell spreading (Bradley et al., 2006). However, arg / fibroblasts have been shown to migrate more rapidly than fibroblasts due to increased actomyosin contractility (Peacock et al., 2007).The critical need for spatial and temporal regulation of Abl kinase activity is highlighted by the recent finding that Abl kinases stimulate Rac induced membrane ruffling in response to adhesion and growth factor signaling, whereas overexpression of Abl inhibits cell spreading in response to fibronectin (Jin and Wang, 2007; Sini et al., 2004). Abl also plays a critical role in the regulation of axon guidance antagonizing repulsive cue signaling by the Roundabout (Robo) receptor (Bashaw et al., 2000) and has more recently been shown to affect adhesion by modulating cross talk between Robo and N cadherin (Rhee et al., 2007; Rhee et al., 2002) Biological function of Abl/Arg In vivo roles of Abl and Arg A role for Drosophila Abl (D abl) in cytoskeletal regulation was initially identified in follicular epithelium: both D abl mutant flies and flies overexpressing high levels of D abl displayed aberrant epithelial morphology with perturbation of the actin 48

62 cytoskeleton (Baum and Perrimon, 2001). D abl has also been implicated in the regulation of adherens junctions. D abl mutants show delayed formation of adherens junctions, decreased accumulation of Armadillo and α catenin at sites of cell cell contact, and impaired dorsal closure during development, which is exacerbated by the additional loss of DE cadherin (Grevengoed et al., 2001). In this regard, D Abl was shown to act at least in part by negatively regulating the subcellular localization of the actin regulator Enabled (Ena). Loss of D Abl results in ectopic Ena and excessive actin accumulation at the apical cortex, whereas ectopic expression of Abl or BcrAbl results in decreased Ena function (Grevengoed et al., 2003; Stevens et al., 2007). Additionally, D Abl mutants display mis localization of other known cytoskeletal regulators including the Arp2/3 complex and the formin Diaphanous, and diaphanous mutations enhance the Abl mutant phenotype (Grevengoed et al., 2003). Data in mice also support a role for Abl family kinases in cytoskeletal reorganization. Abl/Arg double null embryos exhibit delayed closure of the neural tube and defects in the actin cytoskeleton of neurepithelial cells (Koleske et al., 1998). Abl family kinases are critical for development as mice deficient in c Abl and Arg die by embryonic day 11 (E11) and show pervasive apoptosis in all tissues (Koleske et al., 1998). In human leukemias, the kinase domain of Abl is constitutively activated when portions of Abl are fused to Bcr in the Philadelphia chromosomal translocation (Melo, 1996). Moreover, enhanced Arg expression has been 49

63 shown to correlate with metastatic progression in colorectal cancer, and Abl is upregulated in pancreatic ductal carcinoma (Chen et al., 1999; Crnogorac Jurcevic et al., 2002). Abl kinase activity has also recently been shown to be upregulated in human breast cancer cell lines (Srinivasan and Plattner, 2006) Abl kinases regulate response to growth factors and cell migration Abl kinases have been identified as downstream components in several growth factor receptor pathways, including Platelet derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) (Plattner et al., 1999). PDGF stimulation results in transient activation of Abl and Arg, and Abl kinase activity is required for membrane ruffling and proliferation in response to PDGF (Plattner et al., 1999; Plattner and Pendergast, 2003). In this case, phosphorylation of Abl occurs indirectly through Src (Dorey et al., 2001; Plattner et al., 1999). However, whereas Abl is required to induce chemotactic motility in response to PDGF, re expression of Arg fails to rescue the loss of chemotaxis in Abl / Arg / cells providing evidence of distinct roles for the Abl family members (Plattner et al., 2004). Tyrosine phosphorylation of cortactin in response to PDGF stimulation has recently been shown to be Abl dependent and represents one mechanism through which Abl kinases promote the induction of dorsal ruffles (Boyle et al., 2007). Loss of Abl/Arg also impairs Rac activation in response to growth factors, which may be due to decreased tyrosine phosphorylation of the RacGEF Sos 1 (Sini et 50

64 al., 2004). Further, Abl has been shown to be required for tyrosine phosphorylation of WAVE 3 in response to PDGF stimulation, which acts downstream of Rac to promote actin nucleation leading to lamellipodial formation (Sossey Alaoui et al., 2007). The defect in cell proliferation in response to PDGF stimulation in cells lacking Abl kinase activity can also be attributed, at least in part, to inhibition of Rac activation (Boureux et al., 2005). In this regard, cytoplasmic Abl is required for PDGF stimulated induction of Myc and DNA synthesis downstream of a Rac/JNK and a Rac/Nox pathway (Boureux et al., 2005). More recently, Abl kinases have been shown to promote invasion in metastatic breast cancer cells (Srinivasan and Plattner, 2006). While Abl kinases appear to play a positive role in PDGF stimulated cell migration, other evidence points to negative effects on cell motility (Cipres et al., 2007; Frasca et al., 2001; Kain and Klemke, 2001; Plattner et al., 2004). Abl / Arg / MEFs exhibit increased migration, which coincides with decreased tyrosine phosphorylation of Crk at tyrosine 221 and increased Crk/CAS complex formation (Kain and Klemke, 2001). Crk/CAS complex formation has been show to stimulate cell migration (Klemke et al., 1998). HGF stimulation was shown to activate Abl kinase activity in thyroid cancer cells, and treatment with STI571 promoted cell motility in response to HGF treatment (Frasca et al., 2001). Inhibition of Abl kinase activity and overexpression of a Crk mutant lacking the Abl specific tyrosine phosphorylation site resulted in enhanced motility in response 51

65 to HGF in fibroblasts expressing the Met receptor (Cipres et al., 2007). Taken together, these studies suggest that Abl mediated phosphorylation of Crk may act downstream of a diverse set of physiological stimuli to negatively regulate cell migration. These data also point to the importance of cell type and context dependent effects in determining the functional consequence of Abl kinase activation Abl family kinases in intercellular conversations Evidence supporting a critical role for the Abl family of tyrosine kinases in modulating cell signaling downstream of cell cell contact formation is rapidly accumulating. Neuromuscular junction (NMJ) formation is stimulated when agrin release from the nerve induces acetylcholine receptor (AChR) clustering in the postsynaptic muscle (Sanes and Lichtman, 2001). Abl family kinases have been shown to localize to the postsynaptic membrane of the developing NMJ following stimulation of AChRs, and Abl kinase activity was required for agrin induced AChR clustering and enhancement of MuSK tyrosine phosphorylation (Finn et al., 2003). This positions the Abl kinases to participate as mediators of a downstream signaling cascade, which is required to bolster AChR clustering, possibly by participating in a positive feedback loop with the MuSK receptor. The Abl kinases also play an important role in T cell receptor (TCR) signaling following formation of the immunological synapse. Abl kinases are activated following TCR stimulation, and loss of Abl kinase activity results in 52

66 reduced tyrosine phosphorylation of several downstream targets including Zap70, LAT, and PLC γ1 (Zipfel et al., 2004). The physiological consequences of Abl loss of function include decreased IL 2 production and cell proliferation in response to TCR stimulation, again supporting a role for Abl kinases in signal amplification following the engagement of an MHC presenting cell with a T cell (Gu et al., 2007; Zipfel et al., 2004). Mice lacking Abl kinases specifically in T cells are compromised in their ability to mount an immune response to bacterial infection (Gu et al., 2007). The previous examples highlight the role of Abl kinases in normal intercellular signaling, however Abl signaling pathways may also be critical mediators of cell signaling pathways induced during bacterial infection following bacterial cell engagement with normal epithelial cells. During Shigella flexneri infection, Abl kinases are recruited to the cell membrane at sites of bacterial entry and catalytically activated (Burton et al., 2003). Abl kinase inhibition decreases bacterial cell uptake as well as cellto cell spread and inhibits tyrosine phosphorylation of signaling molecules, including Crk and N Wasp, required for these processes (Burton et al., 2005; Burton et al., 2003). Rac activation downstream of Shigella infection was also shown to require Abl kinasemediated phosphorylation of Crk (Burton et al., 2003). Recently, Abl has been shown to be phosphorylated and activated by the bacterial CagA protein following Helicobacter pylori infection (Poppe et al., 2007; Tammer et al., 2007). In this context, Abl kinase 53

67 activity is required for downstream cytoskeletal arrangements leading to enhanced cell motility and sustained phosphorylation of the CagA protein (Poppe et al., 2007; Tammer et al., 2007). Taken together, these data suggest that hijacking of the Abl kinase signaling cascade to promote cytoskeletal re arrangement may be a critical component of signaling from invasive bacteria to invasive host cells. Further, these reports suggest that Abl kinases may function downstream of other receptors to regulate intercellular conversations. 54

68 2. Abl Tyrosine Kinases regulate cell-cell adhesion via Rho GTPAses This chapter appears in modified form in: Proc Natl Acad Sci U S A Nov 6;104(45): Introduction Dynamic regulation of the actin cytoskeleton is required for the formation and dissolution of intercellular adhesions during tissue morphogenesis (Schock and Perrimon, 2002) and pathological processes such as tumor invasion and metastasis (Paszek et al., 2005; Sahai and Marshall, 2002). The formation of cell cell contacts is dependent on the assembly of adherens junctions (Schock and Perrimon, 2002). Adherens junctions are specialized structures that link transmembrane cadherins in neighboring cells to the actin cytoskeleton (Schock and Perrimon, 2002). The extracellular domain of the cadherins cluster in a calcium dependent manner and trigger association of the cadherin cytoplasmic domain with β catenin, which in turn binds to α catenin, a protein that links the cadherin/catenin complex to the actin cytoskeleton (Schock and Perrimon, 2002). Notably, disruption of the actin cytoskeleton, genetic inactivation of actin regulatory proteins, or disrupting the link between the cadherin/catenin complex and the actin cytoskeleton, all result in loss of adherens junctions (Bershadsky, 2004; Ivanov et al., 2005; Vasioukhin et al., 2000). Adherens 55

69 junction formation, stability, and dissolution are processes regulated by Rho family GTPases, which function in part by remodeling the actin cytoskeleton (Thiery, 2002). However, little is known regarding the mechanisms that link cadherin mediated signaling to regulation of Rho GTPases leading to dynamic reorganization of the actin cytoskeleton. Abl (Abl1) and Arg (Abl2) define a family of nonreceptor tyrosine kinases characterized by unique carboxy terminal actin binding domains that can bundle actin (Wang et al., 2001). The Abl kinases regulate a variety of cytoskeletal processes downstream of receptor tyrosine kinases and integrins (Finn et al., 2003; Hernandez et al., 2004; Kain and Klemke, 2001; Moresco et al., 2003; Plattner et al., 1999; Sini et al., 2004). We have shown that Abl kinases play a role in the regulation of intercellular signals at the neuromuscular junction (NMJ) and at sites of contact between invading bacterial pathogens and mammalian host cells (Burton et al., 2003; Finn et al., 2003). Genetic studies in Drosophila and mice support a role for Abl kinases in the regulation of epithelial morphogenesis (Grevengoed et al., 2003; Grevengoed et al., 2001; Koleske et al., 1998). Mice lacking Abl and Arg die before embryonic day 11 and display collapse of the neural tube, which is secondary to disruption of the apical actin latticework in neuroepithelial cells (Koleske et al., 1998). Based on the unique properties of the Abl kinases we asked whether Abl and Arg regulate the formation and/or maintenance of 56

70 adherens junctions in mammalian cells, and sought to define a pathway whereby the Abl kinases modulate these processes. Here we identify a novel role for Abl kinases in the regulation of cell cell adhesion via Rho family GTPases. 2.2 Results Abl Tyrosine Kinases are Required for Cell-Cell Adhesion To determine whether the Abl kinases are involved in the regulation of cadherinmediated cell cell adhesion, we first examined the consequences of genetic inactivation of Abl and Arg on the formation of N cadherin dependent cell cell junctions in mouse embryo fibroblasts (MEFs) (Yonemura et al., 1995). Abl / Arg / MEFs were reengineered to re express Abl and Arg (Burton et al., 2003; Plattner et al., 1999). Cells expressing Abl and Arg displayed continuous cell cell junctions similar to wild type (WT) MEFs as visualized by staining for N cadherin, β catenin and α catenin (Figure 4A, top). In contrast, adherens junction proteins failed to accumulate at sites of cell cell contact in Abl / Arg / MEFs and displayed enhanced cytoplasmic staining (Figure 4A, bottom). Loss of Abl and Arg did not alter the expression levels of adherens junction proteins (Figure 4B). To assess the role of Abl kinases in the formation of adherens junctions, cell monolayers were placed in low calcium media to dissolve pre existing 57

71 A + Abl/Arg N-cadherin β-catenin α-catenin B Abl/Arg Null cells: N-cadherin β-catenin vector +Abl/Arg α-catenin Abl/Arg vector β-tubulin C Low calcium 30 min + calcium 4 h + calcium vector + Abl/Arg Figure 4: Loss of Abl family kinases in fibroblasts disrupts N cadherin based adhesion. Figure 4: Abl/Arg null MEFs transduced with retroviruses encoding Abl and Arg or vector alone were grown to near confluency and either fixed and stained (A) with antibodies to N cadherin, β catenin, or α catenin (red) and counterstained with DAPI (blue) or lysed (B) to evaluate adherens junction protein expression by blotting with the indicated antibodies. (C) Abl/Arg or vector reconstituted null MEFs were grown to subconfluency and subjected to calcium switch to stimulate adherens junction formation. Cells were fixed and stained with antibody against N cadherin (red) and counterstained with DAPI (blue). 58

72 cell cell junctions and then switched to high calcium media to activate cadherinmediated intercellular adhesion. Greater than 90% of cells expressing Abl/Arg formed adherens junctions after 4 hours in high calcium media (Figure 4C, top). In contrast, Abl / Arg / cells displayed a disorganized N cadherin staining pattern and were unable to form discrete adherens junctions even 4 hours after addition of high calcium medium (Figure 4C, bottom). Taken together, these data reveal that Abl kinases are required for the formation of adherens junctions in fibroblasts. We next examined whether Abl kinase activity plays a role in the regulation of epithelial cell cell junctions. Recently confluent NBT II rat epithelial cells were stimulated to form adherens junctions by calcium switch in the presence or absence of STI571, a pharmacological inhibitor of the Abl kinases (Kantarjian et al., 2002; Okuda et al., 2001). In control cells, β catenin accumulated at sites of cell cell contact after incubation in high calcium media (Figure 5A, top). In contrast, cells treated with the Abl kinase inhibitor failed to induce β catenin containing protrusions and to form adherens junctions (Figure 5A, bottom). Abl kinases were markedly inhibited by 10μM STI571 as determined by the reduction of endogenous CrkL phosphorylation on Y207, the Ablspecific site (Figure 5B). To determine whether Abl and Arg proteins are required for intercellular adhesion in E cadherin expressing epithelial cells, Abl and Arg expression was downregulated using RNA interference (RNAi) (Figure 5C). NBT II cells 59

73 A Low calcium 8 h + calcium B 10µM STI571: _ 1 h3 h 6 h control IB: pcrkl Y207 IB: CrkL 10µM STI571 C control sirna Abl sirna D β-catenin siglo merge Abl/Arg sirna Control sirna Arg sirna Abl/Arg sirna Figure 5: Inhibition of Abl kinase activity or protein impairs adherens junction formation in epithelial cells. Figure 5: (A) Confluent NBT II cells were subjected to calcium switch in the absence or presence of 10μM STI571. Cells were fixed and stained with antibody against β catenin (red) and counterstained with DAPI (blue). (B) Tyrosine phosphorylation of the endogenous Abl substrate CrkL at the Abl specific site (Y207) is inhibited following treatment with 10μM STI571. (C) Anti β catenin immunostaining of NBT II cells transfected with control or Abl and/or Arg directed sirnas. (D) Anti β catenin immunostaining of MCF 10A cells co transfected with siglo RISC Free sirna (to identify transfected cells) and with control or Abl and Arg directed sirnas. Images of β catenin (green) and siglo oligo (red) were merged to indicate sirna transfected cells. Scale bars in all figures represent 20 μm. 60

74 A E-cadherin siglo merge B α-catenin siglo merge Abl/Arg sirna Abl/Arg sirna Control sirna Control sirna C sirna: Abl/Arg E-cadherin β-catenin α-catenin β-tubulin Control Abl/Arg Figure 6: Loss of Abl/Arg impairs intercellular adhesion. Figure 6: (A) Anti E cadherin immunostaining of MCF 10A cells co transfected with siglo RISC Free sirna and with control or Abl and Arg directed sirnas. Images of E cadherin (green) and siglo oligo (red) were merged to indicate transfected cells. (B) Anti α catenin immunostaining of MCF 10A cells co transfected with siglo RISC Free sirna and with control or Abl and Arg directed sirnas. Images of α catenin (green) and siglo oligo (red) were merged to indicate transfected cells. (C) MCF 10A cells were transfected with control or Abl and Arg directed sirnas and lysed 48 h posttransfection. Adherens junction protein levels were analyzed by Western blotting with the indicated antibodies. 61

75 transfected with Abl, Arg, or both Abl and Arg directed sirnas showed decreased levels of β catenin containing cell cell junctions. To determine whether this effect is observed in other E cadherin expressing epithelial cells, Abl and Arg expression was downregulated by RNAi in MCF 10A epithelial cells (Figure 5D and 6C). MCF 10A cells transfected with Abl/Arg directed sirnas showed decreased levels of E cadherin and catenins at sites of cell cell contact or lacked cell cell contacts completely with no change in adherens junction protein expression (Figure 5D and 6). Taken together, these data support a critical role for Abl family protein and kinase activity in the formation of epithelial adherens junctions Abl Tyrosine Kinases are Required for the Maintenance of Adherens Junctions Adherens junctions in epithelial sheets are dynamic structures, which undergo constant remodeling (Yamada et al., 2005). To test the involvement of Abl kinases in adherens junction stability, Abl kinase activity was inhibited with STI571 after formation of confluent monolayers of NBT II epithelial cells. Treatment with STI571 for 3 hours resulted in relocalization of β catenin from adherens junctions to the cytoplasm (Figure 7A). After 6 hours of STI571 treatment, only 40% of the cells retained adherens junctions as indicated by reduced β catenin staining at sites of cell cell contact (Figure 7A, B). Adherens junction protein expression was unaffected by Abl kinase inhibition for up to 62

76 A STI571: β-catenin 0 h 3 h 6 h B Cells with adherens junctions (%) STI571: * - 6 h C STI571: E-cadherin β-catenin α-catenin actin - 6 h D STI571: E-cadherin α-catenin 0 h 1 h 3 h 6 h E STI571: α-catenin E-cadherin β-tubulin F STI571: α-catenin - 1 h3 h6 h IP: E-cadherin - 1 h 3 h 6 h E-cadherin Figure 7: Inhibition of Abl kinase activity impairs accumulation of E cadherincatenin complexes at cell cell contacts. 63

77 Figure 7: (A) Confluent NBT II cells were treated with 10μM STI571 for the indicated times, fixed and stained for β catenin (red) and counterstained with DAPI (blue). (B) β catenin staining at sites of cell cell contact was analyzed in 4 independent experiments. *, significant decrease (p=.0004) in adherens junctions in STI571 treated cells versus controls. (C) Recently confluent NBT II cells were untreated or treated with 10μM STI571 for 6 h. Total lysates were examined by immunoblotting with antibodies against the indicated adherens junction proteins. (D) Confluent MCF 10A cells were grown to confluence and treated with 10μM STI571 for the indicated times. Cells were fixed and stained for the indicated adherens junction proteins. (E) Confluent MCF 10A cells were untreated or treated with 10μM STI571 for the indicated times. Total lysates were examined by immunoblotting with antibodies against the indicated adherens junction proteins. (F) To evaluate integrity of the cadherin catenin complex in response to Abl kinase inhibition, the level of α catenin bound to E cadherin was assessed by immunoprecipitation of E cadherin from 500μg lysate and immunoblotting with an antibody against α catenin. Blots were stripped and reprobed with an antibody against E cadherin to demonstrate equal protein levels. 64

78 6 hours (Figure 7C). Similarly, STI571 treatment of MCF 10A cells promoted mislocalization of adherens junction proteins (Figure 7D) with no change in E cadherin and α catenin protein levels (Figure 7E) or the integrity of the E cadherin/α catenin complex (Figure 7F). Moreover, we did not observe any changes in the tyrosine phosphorylation of components of the cadherin/catenin complex in the absence of Abl kinases or after inhibition of Abl kinase activity (data not shown) Cell-cell Adhesion Leads to Abl Kinase Activation and Recruitment to Sites of Cell-Cell Contact To test whether Abl kinases are catalytically activated by cell cell adhesion, we analyzed the phosphorylation of the Abl substrates CrkII or the related CrkL using phosphospecific antibodies against the Abl specific sites, which have been shown to be unphosphorylated in Abl / Arg / MEFs or in cells lacking Abl kinase activity (Burton et al., 2003; Zipfel et al., 2004). Abl kinase activity was upregulated by 5 minutes following calcium switch to induce cadherin mediated cell cell adhesion, and Abl activation was sustained for 30 minutes in WT MEFs (Figure 8A). Similar kinetics were observed in NBT II cells subjected to calcium switch using in vitro kinase assays with GST Crk to measure Abl and Arg kinase activity (Plattner et al., 2003) (Figure 8B). To eliminate the possible contribution of intercellular adhesion independent effects, we compared Abl activation in sparse versus confluent cultures and found that Abl kinase activity is 65

79 A + Ca 2+ + EGTA 5 min 30 min IB: pcrk Y221 Fold: IB: Crk B IP: Arg Fold: IP: Abl Fold: IB: Abl 0 min + Ca 2+ 1 min 2 min 5 min 10 min 30 min 60 min C Ca 2+: IB : pcrkl Y207 Fold: Confluent cultures Sparse cultures Hi Low Hi Hi Hi Low Hi Hi 5 m 15 m 5 m 15 m D * * IB : CrkL min 5 min 15 min confluent cultures sparse cultures Figure 8: Cell cell adhesion regulates the activity of endogenous Abl kinases. Figure 8: (A) Activation of Abl and Arg kinases in response to calcium switch in wildtype (WT) MEFs was assessed by immunoprecipitation of Crk and immunoblotting with a phosphospecific antibody to Y221. Blots were stripped and reprobed with an anti Crk antibody to demonstrate equal loading. Results shown are representative of 3 independent experiments. (B) Confluent NBT II epithelial cells were subjected to calcium switch and Abl or Arg protein was immunoprecipitated as indicated using specific antibodies, and in vitro kinase assays were performed using GST Crk as substrate. Abl/Arg protein expression in whole cell lysates (bottom panel) was assessed by immunoblotting with 8E9 monoclonal antibody. (C) Abl/Arg kinase activity in response to calcium switch was assayed in confluent versus sparse epithelial cell cultures. (D) Abl kinase activity was quantitated using densitometry with pcrkl levels indexed against low calcium for each condition. Results represent three independent experiments. *, indicates p< Fold changes in pcrk or pcrkl levels are indicated below the corresponding lanes. 66

80 unaltered in the absence or presence of calcium in sparse cultures (Figure 8C, D). In contrast, Abl kinase activity increases in a time dependent manner after calcium switch in confluent cell monolayers (Figure 8C, D). Thus, the regulation of Abl kinase activity after calcium switch is dependent on cadherin mediated cell cell contacts. To determine whether Abl and Arg are recruited to sites of cell cell contact to participate in signaling, we generated MDCK cell lines expressing GFP tagged versions of the Abl kinases and examined the localization of these kinases during adherens junction formation. Arg accumulated at sites of cell cell contact and in the cytoplasm in both confluent cells and in cells stimulated to re form adherens junctions by switch to high calcium medium (Figure 9A). Similar results were observed for GFP Abl (data not shown). In confluent MCF 10A cells, Arg also co localized with β catenin at sites of cellcell contact (Figure 9C), and E cadherin and β catenin co immunoprecipitated with GFP Arg (Figure 9B). Disruption of cadherin mediated cell cell adhesion (low calcium) resulted in re distribution of Arg from adherens junctions to the cytoplasm (Figure 9A, C). Following calcium switch to induce adherens junction formation, GFP Arg relocalized in lamellipodial extensions and at sites of nascent cell cell contacts (Figure 9C). Together, these data further support a role for Abl and Arg in adherens junction regulation as both proteins are recruited to sites of cell cell contact, are associated with 67

81 A Hi calcium Low calcium 15 min + Hi calcium B Vector GFP-Arg IP: GFP E-cadherin β-catenin GFP C Hi calcium Low calcium 1 h + Hi calcium 6 h + Hi calcium merge GFP β-catenin Figure 9: Arg localizes to adherens junctions. Figure 9: (A) MDCK cells expressing GFP tagged Arg were seeded onto glass coverslips, grown to confluence, and subjected to calcium switch. Cells were fixed and GFP fluorescence examined by microscopy. (B) MCF 10A cells expressing vector or GFPtagged Arg were grown to confluence and lysed in 1% Triton X 100 lysis buffer. Anti GFP antibody was used to immunoprecipitate Arg from 1mg pre cleared lysate, followed by immunoblotting for E cadherin or β catenin. Blots were stripped and reprobed for GFP. (C) MCF 10A cells expressing GFP tagged Arg were seeded onto glass coverslips, grown to confluence, and subjected to calcium switch. Cells were fixed and stained for β catenin and GFP. Images of β catenin (red) and GFP (green) were merged to indicate co localization (yellow). 68

82 E cadherin/β catenin complexes and are activated in epithelial cells in response to cadherin engagement Abl Kinases Regulate Rac Activation in Response to Cadherin Engagement via Crk/CrkL To test whether Abl kinases are sufficient to promote adherens junction formation, we expressed a constitutively active mutant form of Arg (ArgPP) (Plattner et al., 2004) in HeLa cells. These cells express N cadherin and exhibit weak, immature adherens junctions. ArgPP expression resulted in enhanced, continuous staining of β catenin at sites of cell cell contact (Figure 10A). Thus, Abl kinases are required for adherens junction formation and promote N cadherin mediated adhesion in cells with relatively weak junctions. Because formation of cell cell junctions correlates with increased phosphorylation of Crk/CrkL proteins at the Abl specific sites, we next examined whether Crk family proteins play a role in ArgPP induced adherens junction strengthening by analyzing the consequences of Crk/CrkL sirna mediated knockdown. We observed a marked reduction in the integrity of adherens junctions in ArgPPexpressing cells transfected with Crk and CrkL sirnas (Figure 10B, C). Similarly, adherens junction integrity was markedly impaired in NBT II epithelial cells transfected 69

83 A vector ArgPP B vector ArgPP Crk/CrkL sirna Control sirna C sirna: Crk CrkL β-tubulin control Crk/CrkL D control sirna Crk/CrkL sirna Figure 10: Crk/CrkL function downstream of Abl mediated adherens junction formation in epithelial cells. Figure 10: (A) β catenin localization was visualized in vector or ArgPP expressing HeLa cells. Expression of ArgPP results in enhanced, continuous staining of β catenin at sites of cell cell contact. (B) β catenin staining in vector or ArgPP expressing HeLa cells transfected with control or Crk and CrkL directed sirna oligos. (C) The levels of Crk/CrkL proteins in lysates from HeLa cells transfected with control or Crk/CrkLspecific sirnas were analyzed by blotting with the indicated antibodies; β tubulin was used as a loading control. (D) β catenin staining in NBT II cells transfected with control or Crk and CrkL directed sirna oligos. 70

84 with Crk and CrkL sirnas (Figure 10D). Taken together these data reveal a critical role for the Abl kinases in adherens junction formation in mammalian cells and suggest that this effect may be mediated in part by Crk proteins. The Rac GTPase has been implicated in the formation of adherens junctions (Ehrlich et al., 2002; Noren et al., 2001). In agreement with previous findings (Noren et al., 2001), we observed that Rac was activated by 5 min of cadherin engagement (Figure 11A). Basal levels of activated Rac were unchanged in confluent monolayers of WT or Abl / Arg / MEFs grown in complete calcium rich media (data not shown). Significantly, Rac activation induced by adherens junction formation was delayed and markedly reduced in Abl / Arg / cells (Figure 11A, B), and in WT MEFs subjected to calcium switch in the presence of the Abl kinase inhibitor STI571 (Figure 11C). Previous studies have linked tyrosine phosphorylation of Crk to Rac activation following bacterial infection and integrin engagement (Abassi and Vuori, 2002; Burton et al., 2003). We found that overexpression of a Crk mutant lacking the Abl specific phosphorylation site in MEFs resulted in decreased Rac activation in response to calcium switch during adherens junction formation (Figure 11D, E). The inhibitory effect of the Crk mutant was similar to that induced by Abl kinase inhibition. Taken together, these data suggest that Abl mediated phosphorylation of Crk is required for maximal activation of Rac downstream of cadherin engagement. 71

85 A control Abl/Arg null Minutes: Rac-GTP Fold: Total Rac Rac-GTP Fold: Total Rac B * 0 min 5 min 15 min Control Null C Rac-GTP Minutes: control Fold: STI Fold: D control CrkYF Minutes: Rac-GTP Fold: Total Rac Rac-GTP Fold: Total Rac E * * 0 min 5 min 15 min Control CrkYF Figure 11: Abl kinases regulate Rac activation in response to cadherin engagement. Figure 11: (A) Rac activation in serum starved WT or Abl / Arg / MEFs grown to confluency and subjected to calcium switch. (B) Rac activation was quantitated using densitometry with Rac GTP levels indexed against low calcium in each condition. Results represent three independent experiments. *, indicates p< (C) Rac activation in serum starved WT or STI571 treated MEFs grown to confluency and subjected to calcium switch. (D) Rac activation in serum starved WT MEFs expressing vector (control) or CrkYF was analyzed in cells grown to confluency and subjected to calcium switch. (E) Rac activation was quantitated using densitometry with Rac GTP levels indexed against low calcium in each condition. Results represent four independent experiments. *, indicates p<

86 2.2.5 Abl Kinases Regulate the Architecture of the Actin Cytoskeleton in Epithelial Cell Sheets via the Rho-Rock Pathway The Abl kinases regulate multiple cytoskeletal processes such as membrane ruffling, chemotaxis, and cell spreading (Pendergast, 2002). Dynamic regulation of the actin cytoskeleton is required for the formation, stability, and dissolution of adherens junctions (Ivanov et al., 2005; Pendergast, 2002; Vaezi et al., 2002; Vasioukhin et al., 2000). Therefore, we tested whether Abl/Arg kinase inhibition could alter the cytoskeletal architecture of recently confluent cells. Epithelial cell clusters display thick cortical bundles of actin at cell cell borders (Figure 12A). In contrast, cells treated with the Abl kinase inhibitor showed decreased cortical actin, increased stress fiber formation, accompanied by the appearance of gaps between cells, and increased numbers of spiky membrane protrusions over time (Figure 12A). The enhanced formation of stress fibers in STI571 treated cells suggested that inhibition of Abl family kinases may result in increased Rho activity. Indeed, cellular levels of Rho GTP increased following STI571 treatment, which correlated with the appearance of stress fibers (Figure 12A, B, C). To determine whether loss of Abl and Arg protein could similarly affect cadherin mediated regulation of Rho activity, we examined Rho activation in epithelial cells where Abl and Arg proteins were knocked down by RNAi. In agreement with previous findings (Noren et al., 2001), cadherin engagement 73

87 A STI571: 0 h 3 h 6 h actin D STI571: 0 h 1 h 3 h pmlc B STI571 (h): Rho-GTP Fold-change Total Rho C * h 0.5 h 1h 3 h E Control sirna Rho-GTP Total Rho Hi Low Hi Ca 2+ Ca 2+ Ca 2+ 5 min Fold-change F * Abl/Arg sirna Rho-GTP Total Rho Fold-change min 5 min control sirna Abl/Arg sirna Figure 12: Abl kinases regulate the actin cytoskeleton and Rho activity in epithelial cell monolayers. Figure 12: Recently confluent NBT II cells were treated with 10μM STI571 for the indicated times. (A) Staining with AlexaFluor 488 conjugated phalloidin (green) and counterstained with DAPI (blue). (B) Lysates were analyzed for Rho activity using the EZ Detect Rho activation kit. Total Rho protein levels were analyzed by immunoblotting. (C) Rho activity was quantitated using densitometry with Rho levels indexed against the untreated condition. Results represent three independent experiments. *, indicates p< (D) Cells were fixed and stained with a phosphospecific antibody to pmlc (red) and counterstained with DAPI (blue). (E) MCF 10A cells were transfected with control or Abl and Arg directed sirna oligos, grown to confluence, and serum starved overnight. Cells were subjected to calcium switch and Rho activity was assayed activity using the EZ Detect Rho activation kit. (F) Rho activity was quantitated using densitometry with Rho levels indexed against the untreated condition. Results represent three independent experiments. *, indicates p<

88 following switch from low to high calcium suppressed Rho activity in control epithelial cells (Figure 12E, F). In contrast, Rho inhibition following cadherin engagement was not observed in the Abl/Arg sirna treated confluent monolayers (Figure 12E, F). Thus, the Abl family kinases are required for cadherin dependent inhibition of Rho activity. Rho activation may result in increased acto myosin contractility mediated through phosphorylation of the myosin regulatory light chain (MLC) of myosin II by Rho activated kinases (Bresnick, 1999). Inhibition of Abl family kinases resulted in a time dependent increase of MLC phosphorylation and enhanced accumulation of phospho MLC at the cell periphery, which correlated with increased formation of stress fibers (Figure 12D). Similarly, Abl kinase inhibition resulted in enhanced phosphorylation of MLC as assessed by immunoblotting to detect phospho serine 19 of MLC (de Rooij et al., 2005) (data not shown). Rho signaling has been shown to regulate both the formation and dissolution of adherens junctions, and activation of the Rho effector ROCK has been shown to disrupt adherens junctions in epithelial cells (Avizienyte et al., 2004; Bhowmick et al., 2001; Braga et al., 1999; Paszek et al., 2005; Sahai and Marshall, 2002; Shewan et al., 2005; Vaezi et al., 2002). To test whether the observed effects of Abl/Arg kinase inhibition are dependent on ROCK kinase activity, we examined whether the phenotypes induced by loss of Abl kinase activity could be reversed by inhibition of ROCK. Consistent with 75

89 previous findings (Sahai and Marshall, 2002), cells treated with the ROCK kinase inhibitor Y27632 for 1 hour did not display adherens junctions defects, but did show a marked decrease of stress fibers at the cell center (Figure 13A). Significantly, cells treated with both STI571 and Y27632 failed to display any of the aberrant morphology characteristic of STI571 treated cells, with β catenin and actin remaining localized to sites of cell cell contacts; also, stress fiber formation was inhibited in these cells (Figure 13A, B, C). Notably, whereas inhibition of the Abl kinases resulted in a 60% decrease in the percentage of cells with mature, continuous adherens junctions, this effect was blocked by inhibition of ROCK (Figure 13C). Further, dual inhibition of both Abl and ROCK kinases abolished the increase in pmlc and the enhanced localization of MLC protein to the cell membrane (data not shown). Thus, inhibition of Abl kinase activity leads to upregulation of Rho ROCK signaling, enhanced MLC phosphorylation, and increased acto myosin contractility, thereby promoting the dissolution of cell cell adhesions. Together, these data support a model whereby endogenous Abl kinases normally function to modulate Rho ROCK activity and maintain cell cell contacts in epithelial cell sheets (Figure 14). 76

90 A control actin β-catenin B Cells with stress fibers (%) * * */** 10µM STI571 C control STI571 Y27632 STI571 + Y µM Y µM STI µM Y27632 Cells with mature cell-cell junctions (β-catenin) * control STI571 Y27632 STI571 + Y27632 * Figure 13: Disruption of adherens junctions in response to inhibition of Abl kinase activity is reversed by inhibition of ROCK kinase activity. Figure 13: (A) Confluent NBT II cells were treated with 10μM STI571, 10μM Y27632, or both STI571 and Y27632 for 1 hour, fixed and stained with phalloidin (green) or anti βcatenin (red), and counterstained with DAPI (blue). (B) Stress fiber formation was analyzed by examining phalloidin staining. Results shown represent the mean of 4 independent experiments. *, significant increase (control vs. STI571 treatment p<.001) or decrease (control vs. Y27632 treatment p<.05, control vs. STI571 + Y27632 treatment p<.05) in stress fibers. **, significant decrease (p<.001) in stress fibers in cells treated with both STI571 and Y27632 together compared to STI571 treated cells. (C) The number of cells with mature adherens junctions marked by continuous β catenin staining at sites of cell cell contact was quantified in 4 independent experiments, in which 9 random fields were examined per condition. *, significant decrease (p<.001) or increase (p<.001) in adherens junctions in cells treated with STI571 in the absence or presence of Y

91 α β P120 catenin α - catenin β - catenin P120 catenin Cadherin P120 cat. β β - cat. α α - cat. Rho-GDP? RhoGAP Y P Abl? F Actin Rho-GTP Crk/ CrkL P Y? ROCK P Rac-GDP MLC RacGAP RacGEF P Rac-GTP ROS Enhanced Contractility RhoGEF Figure 14: Model for Abl family kinase regulation of adherens junctions. 78

92 2.3 Discussion Here we identify the Abl family of tyrosine kinases as critical mediators of cadherin dependent adhesion in mammalian cells through regulation of Rho family GTPases. We have employed genetic ablation and pharmacological inhibition to show that Abl kinases are required for the formation of adherens junctions and that loss of Abl kinases impairs Rac activation induced by cadherin engagement. Additionally, we show that Abl gain of function promotes adherens junction formation in cells with weak junctions and that Crk/CrkL proteins are required for this strengthening phenotype. Significantly, we show for the first time that endogenous Abl kinases are activated by cadherin engagement. We found that Abl and Arg are recruited to nascent junctions and are present in E cadherin/β catenin protein complexes. Additionally, we have shown that Abl kinase activity is required for the maintenance of adherens junctions in epithelial cell sheets, as inhibition of these kinases results in the redistribution of adherens junction components from sites of cell cell contact to the cytoplasm. Abl kinases are not only required for maximal Rac activation induced by cadherin mediated adhesion, but are likely to modulate cell cell adhesion via regulation of other pathways. In this regard, we have shown that Abl mediated regulation of the Rho ROCK myosin signaling pathway is essential for adherens junction stability. Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its 79

93 downstream target ROCK, leading to enhanced phosphorylation of the myosin II regulatory light chain. Activation of this signaling pathway leads to enhanced stress fiber formation and increased acto myosin contractility, thereby disrupting adherens junctions. These findings identify a novel role for the Abl tyrosine kinases as regulators of intercellular adhesion through their ability to modulate both Rac and Rho GTPasedependent pathways. Notably, we showed that Abl kinases activated by cadherin engagement phosphorylate Crk/CrkL proteins, and that both Abl and Crk family proteins are required for adherens junction integrity in a pathway upstream of Rac. We and others have shown that Crk phosphorylation on the Abl specific site is required for Rac activation and membrane localization [Figure 11E, (Abassi and Vuori, 2002; Burton et al., 2003)]. The mechanism by which Crk phosphorylation by Abl results in Rac activation may involve the recruitment of the Crk binding protein DOCK180, a guanine nucleotide exchange factor (GEF) for Rac (Kiyokawa et al., 1998). Alternatively, Abl may activate Rac by phosphorylating Sos 1, which was reported to stimulate the Rac GEF activity of Sos 1 (Sini et al., 2004). Abl kinases are required for both activation of Rac and inhibition of Rho at adherens junctions, and it is possible that these events may be coordinately linked. Abl may downregulate Rho signaling indirectly by activating a Rac ROS pathway (Nimnual 80

94 et al., 2003), or by directly regulating the activities of a RhoGAP or a RhoGEF. In this regard, activated RacV12 was reported to inhibit Rho via p190 RhoGAP (Nimnual et al., 2003), and p190 RhoGAP was shown to be phosphorylated in an Arg dependent manner in response to integrin engagement in neuronal cells (Hernandez et al., 2004). However, we have not detected any changes in p190 RhoGAP tyrosine phosphorylation in the absence of Abl/Arg kinases or upon STI571 treatment in epithelial cells or fibroblasts (data not shown). Nor did we observe any changes in complex formation between p190 RhoGAP and either p120 RasGAP, which is required for activating p190 RhoGAP activity, or p120 catenin (data not shown). Moreover, in agreement with published data (Wildenberg et al., 2006), we found that p190 RhoGAP does not localize to adherens junctions in the cells analyzed here (data not shown). Thus, our data suggest that Abl substrates other than p190 RhoGAP are likely to regulate adherens junction formation and maintenance. We have however, identified the Crk/CrkL family of adaptors as critical players in the Abl mediated regulation of adherens junctions. Future studies are needed to identify all the GEFs and GAPs involved in the regulation of Rac and Rho during adherens junction formation and stability in epithelial cells and fibroblasts. Together our findings implicate the Abl family of tyrosine kinases as regulators of intercellular adhesion in epithelial cells and fibroblasts, and support the model depicted in Figure 14. These data and our previous results demonstrating that Abl 81

95 kinases are required for the formation of the neuromuscular junction, suggest that Abl kinases may function to modulate adhesive interactions between the same or different cell types. Changes in the activation of the Abl kinases may also modulate cell cell adhesion during tumor progression and metastasis. In this regard, enhanced expression of Abl or Arg has been reported in a subset of metastatic colon carcinomas, pancreatic ductal carcinoma, and renal medullary carcinoma (Chen et al., 1999; Crnogorac Jurcevic et al., 2002; Simpson et al., 2005). Excessive Abl/Arg kinase activity may disturb the proper balance of Rho and Rac activation and induce altered cytoskeletal reorganization, thereby affecting cell cell adhesion. 82

96 3. A closer look at Abl-Crk-Rac 3.1 Background While many of the players in the regulation of adherens junctions have been identified, their individual roles and the way they interact with one another is often controversial. This is particularly true for regulators of the actin cytoskeleton. For example, activation of Rac, Rho, and Src kinases have all been implicated in both the formation and dissolution of adherens junctions (Calautti et al., 1998; Lilien and Balsamo, 2005; Lozano et al., 2003; Sahai and Marshall, 2002; Yap and Kovacs, 2003). The biological consequence of Rho signalling is thought to be determined by the choice of downstream effector: activation of Dia promotes intercellular adhesion, while activation of ROCK disrupts adhesion (Sahai and Marshall, 2002). However little is known regarding the mechanism through which specific effectors are targeted. Another unresolved question in adherens junction biology is how signaling to promote actin polymerization is sustained throughout the process of junction formation. Rac is activated following cadherin engagement and initially required to promote actin polymerization, which may also facilitate cadherin clustering (Brunton et al., 2004). Initial cell cell contacts resemble spots of adhesion and the process through which the membranes are sealed with adherens junctions has been likened to zippering. Actin 83

97 polymerization is also required for this closing of the adhesion zipper, and, in fact, Rac activation has been shown to occur in a biphasic manner downstream of cadherin engagement (Noren et al., 2001). Recently, it was shown that rounds of Rac activation and downregulation occur at the periphery of the contacting membranes between neighboring cells in the process of epithelial cell cell adhesion (Yamada and Nelson, 2007). While it is possible that Rac is activated initially through one mechanism and later activated again, it is also possible that Rac activation stimulates a positive feedback loop, which is required for sustained signaling and actin polymerization during the maturation of adherens junctions. The Abl family of tyrosine kinases, Abl and Arg, are known to have a role in modulating cytoskeletal responses (Pendergast, 2002). Abl and Arg participate in signaling events leading to actin polymerization downstream of integrin engagement, growth factor receptor signaling, bacterial infection, and repulsive cues in axon guidance (Bashaw et al., 2000; Bradley et al., 2006; Burton et al., 2003; Plattner et al., 1999; Sini et al., 2004). We recently showed for the first time that Abl kinases are activated downstream of cadherin engagement and recruited to nascent cell cell contacts in mammalian cells (Zandy et al., 2007). In this regard, Abl kinases are required for maximal Rac activation following the stimulation of cell cell adhesion (Zandy et al., 2007). This process is dependent on the Abl substrate Crk, which becomes tyrosine 84

98 phosphorylated during the formation of adherens junctions (Zandy et al., 2007). Likewise, a requirement for Crk in Rac activation has been identified in regulation of other cellular processes including signaling downstream of Shigella infection and the Met receptor (Burton et al., 2003; Lamorte et al., 2002b). 3.2 Results We previously showed that Abl kinases are sufficient to promote adherens junction formation by expressing a constitutively active mutant form of Arg (Arg PP) (Plattner et al., 2004) in HeLa cells (Zandy et al., 2007). ArgPP expression resulted in enhanced, continuous staining of β catenin at sites of cell cell contact. Similarly, overexpression of activated Rac has been shown to promote adherens junction formation in some epithelial cells (Wildenberg et al., 2006). Indeed, overexpression of constitutively active Rac (RacV12) in HeLa cells phenocopied the effect of active Arg expression. Thus, activated forms of both Abl kinases and Rac promote N cadherinmediated adhesion in cells with relatively weak junctions (Figure 15A). We showed that adherens junction strengthening in ArgPP expressing cells required the presence of the Crk family proteins, Crk and CrkL (Zandy et al., 2007). To determine whether Crk family proteins also play a role in RacV12 induced adherens junction strengthening, we examined the consequence of Crk/CrkL sirna mediated knockdown. We observed a marked reduction of adherens junctions in both RacV12 and ArgPP expressing cells 85

99 transfected with Crk and CrkL sirnas (Figure 15A). These data reveal a critical role for the Crk family of adaptors in adherens junction formation in mammalian cells and suggest that they act downstream of both Abl kinases and Rac. Because expression of RacV12 phenocopied the effect of ArgPP expression in HeLa cells and Crk proteins were required downstream of both activated Rac and Abl for strengthening of adherens junctions. We next asked whether the Abl kinases may be required for RacV12 induced enhancement of adherens junctions. To determine whether Abl kinases are required for this phenotype, we examined the consequence of Abl/Arg knockdown in HeLa cells expressing RacV12. Depletion of Abl/Arg or treatment with the Abl kinase inhibitor STI571 (Gleevec) decreased the RacV12 enhanced localization of β catenin to sites of cell cell contact (Figure 15B, data not shown). Expression of RacV12 led to increased phosphorylation of the Abl substrate CrkL, which was reduced in cells with downregulated Abl/Arg protein (Figure 15C) or cells treated with STI571 (data not shown) suggesting that enhanced Abl signaling through Crk/CrkL family proteins may play a role in the observed phenotype. Expression of constitutively active RacV12 has been shown to induce production of reactive oxygen species (ROS) (Nimnual et al., 2003). Interestingly, ROS is a potent activator of the Abl kinases, and promotes enhanced phosphorylation of Crk (Sun et al., 2000a). Taken together, these data suggest that Crk family proteins may promote 86

100 A vector ArgPP RacV12 Crk/CrkL sirna Control sirna B sirna: control Abl/Arg C vector RacV12 vector RacV12 sirna: Abl/Arg β-tubulin pcrkl Y207 CrkL control Abl/Arg control Abl/Arg Figure 15: Abl and Crk function downstream of Rac activation. Figure 15: (A) β catenin staining in vector, RacV12, or ArgPP expressing HeLa cells transfected with control or Crk and CrkL directed sirna oligos. (B) β catenin staining was examined in vector or RacV12 expressing HeLa cells transfected with control or Abl and Arg directed sirna oligos. (C) HeLa cells were lysed 48 h post transfection. Abl/Arg protein levels and CrkL phosphorylation and expression were examined using Western blotting. 87

101 adherens junction formation and strengthening downstream of active Rac and Abl kinases. Whereas our data suggest a positive role for Crk in regulating intercellular adhesion, overexpression of Crk has been shown to promote the dissolution of adherens junctions in other contexts (Lamorte et al., 2002a; Lamorte et al., 2002b; Suzuki et al., 2005). We reasoned that tyrosine phosphorylation of Crk may hold the key to resolving this paradox. We observed that enhanced phosphorylation of Crk downstream of ArgPP and RacV12 expression was correlated with adherens junction strengthening. If Crk overexpression led to an increase in unphosphorylated Crk levels due to limiting amounts of active Abl kinases, the prediction is that unphosphorylated Crk may promote the dissolution of cell cell junctions, and that perhaps overexpression of ArgPP might rescue the inhibitory effect of Crk overexpression on cell cell adhesion. To test this hypothesis, we overexpressed Crk in NBT II cells and, indeed, observed reduced β catenin staining at sites of cell cell contact and enhanced cell spreading, which phenocopied the effect of STI571 inhbition of Abl kinase activity (Figure 16). In contrast, cells co expressing Crk and ArgPP retained β catenin at adherens junctions and showed reduced cell spreading (Figure 16). These data support our hypothesis that Argmediated tyrosine phosphorylation of Crk promotes intercellular adhesion, while unphosphorylated Crk promotes the destabilization of intercellular adhesions. 88

102 vector ArgPP vector Crk Figure 16: ArgPP expression rescues negative effects of Crk overexpression on adherens junctions. Figure 16: β catenin staining in vector or Crk expressing NBT II cells, co expressing vector or ArgPP. 89

103 3.3 Discussion We showed that Abl kinases activated by cadherin engagement phosphorylate Crk/CrkL proteins and function upstream of Rac (Zandy et al., 2007). We and others have shown that Crk phosphorylation on the Abl specific site is required for Rac activation and membrane localization (Abassi and Vuori, 2002; Burton et al., 2003; Zandy et al., 2007). The functional significance of Crk phosphorylation during adherens junction formation is likely due to its role in regulating Rac activity, possibly through regulating its localization to sites of cell cell contact. However, the fact that Rac activation also leads to Crk dependent formation of adherens junctions suggest the role of Crk is complex. In isolated fibroblasts, tyrosine phosphorylation has been shown to disrupt Crk/CAS complexes, which stimulate cell migration (Klemke et al., 1998). Conversely, STI571 treatment or Abl/Arg knockdown decrease tyrosine phosphorylation of Crk at tyrosine 221, which may lead to increased Crk/CAS complex formation as in Abl / Arg / fibroblasts (Kain and Klemke, 2001). Future studies are needed to examine how RacV12 and ArgPP expression affect the intermolecular complexes containing Crk and to determine their relevance to adherens junction regulation. Moreover, we discovered that ArgPP expression rescues the destabilizing effect of Crk overexpression on adherens junctions. This suggests that there is a critical difference in the biological activity of phosphorylated vs. unphosphorylated Crk. Our 90

104 results also raise the question of how levels of phosphorylated Crk are measured by the cell: is there an absolute amount of tyrosine phosphorylation of Crk required to promote adherens junction formation? how does the balance of phosphorylated versus unphosphorylated Crk affect adherens junctions? and does Crk phosphorylation lead to changes in Rac/Crk localization or duration of signaling? We have also demonstrated a requirement for Crk/CrkL in RacV12 induced and ArgPP induced strengthening of adherens junctions. We showed that expression of constitutively active Rac leads to increased Abl kinase activity and tyrosine phosphorylation of Crk. Thus, Abl Crk signaling may function not only upstream but also downstream of Rac in a positive feedback loop (Figure 17). Abl activation downstream of cadherin engagement could promote Rac activation and localization to nascent cell cell contacts. Heightened Rac activity could then reinforce the initial wave of Abl kinase activation and sustain Crk phosphorylation, leading to recruitment of more Rac to sites of cell cell contact. This positive feedback loop may be required to generate enough force from actin based lamellipodia to hold the cells together during both the formation and maturation of adherens junction. Follow up studies using fluorescent tagged Arg, Crk, and Rac mutants may shed some light on the hierarchical requirement for each of these molecules in the regulation of one another during adherens junction formation and maintenance. 91

105 α α - catenin β β - catenin Cadherin Rac-GTP ROS (H 2 O 2 ) P Y Crk/ CrkL Abl P Y α β β - cat. α - cat.?? RhoGAP Rho-GDP Decreased contractility & stabilization of cellcell junctions RhoGEF Figure 17: Proposed model for Abl Crk Rac signaling. 92

106 The mechanism through which Rac activation leads to increased Abl kinase activity may be due to generation of ROS in cells. Expression of constitutively active RacV12 promotes production of ROS (Nimnual et al., 2003). Moreover, ROS was reported to activate Abl kinases and to promote tyrosine phosphorylation of Crk (Sun et al., 2000a). Thus, the Rac ROS pathway may strengthen adherens junctions by activating Abl kinases and promoting Crk tyrosine phosphorylation (Figure 17). Generation of ROS is commonly induced in cancer cells through hyperactivation of growth factor signaling pathways and Rac activation. In this regard, hyperactivation of the Met receptor leading to Rac induced generation of ROS is required for the in vivo prometastatic behavior of Met (Ferraro et al., 2006). This raises the intriguing possibility that Abl Crk signaling may play a role in this process as well. Rac activation has been shown to promote both the formation and dissolution of adherens junctions, dependent on cellular context (Lozano et al., 2003). Our novel finding that Abl kinase activity is enhanced in the presence of high levels of Rac activity suggest that Abl kinases may also have opposite effects on adherens junctions. In this regard, high levels of Abl kinase activity have been detected in metastatic vs. nonmetastatic breast cancer cells, and blocking Abl kinase activity impaired invasion (Srinivasan and Plattner, 2006). Similarly, we have observed elevated levels of Abl kinase activity in metastatic vs. non metastatic mouse mammary tumor cell lines (Figure 93

107 18). These data raise the larger question of how the biological consequences of elevated Abl kinase or Rac activity may result in distinct outcomes leading to either formation or dissolution of adherens junctions. Factors involved in this regulation may include integration of various signals, levels of signaling, spatio temporal regulation, and downstream effectors. We have identified a positive feedback loop between Abl Crk Rac in the context of intercellular adhesion, and this feedback loop may function in other cellular processes mediated by Abl and Rac signaling, including cell migration, endocytosis, and processes that require cytoskeletal reorganization. 94

108 A B IP: CrkL IB: Abl/Arg IB: β-tubulin 4T1 4TO7 66c14 67NR 4T1 66c14 4TO7 67NR IB: pcrk IB: CrkL Figure 18: Hyperactivation of Abl kinase activity in metastatic breast cancer cell lines. Figure 18: Mouse mammary tumor cell lines 4T1, 4TO7, 66c14, and 67NR were grown to similar confluency (70%) and lysed in 1% Trition lysis buffer in the presence of protease and phosphatase inhibitors. (A) To assess Abl/Arg activity levels in each cell type, lysates were immunoprecipitated with antibody to the Abl substrate CrkL followed by immunoblotting with a phospho Crk antibody which recognizes CrkL Y207, the Ablspecific site. (B) Total levels of Abl/Arg protein are shown and β tubulin is shown as a loading control. Abl kinase activity is lowest in the non metastatic line 67NR and highest in the metastatic cell line 4T1. Interestingly, Abl kinase activity is higher in the 4T1 cell line than in the 66c14 cell line. Unlike the 66c14 cells, the 4T1 cells metastasize in the absence of lymphatics. Abl family kinases may play a role in this process. 95

109 4. Role for Abl kinases downstream of the Met receptor 4.1 Background Dynamic remodeling of cell cell adhesions is critical for morphogenetic changes that occur during normal development and is also required for tumor invasion and metastasis (Thiery, 2002). Hepatocyte growth factor (HGF) was first characterized as a physiological factor promoting cell scattering remodeling adherens junctions leading to dissociation of intercellular contacts (Birchmeier et al., 2003; Stoker et al., 1987). Subsequently, HGF has been identified as a critical mediator of tumor progression in various epithelial derived tumors (Hurle et al., 2005; Jiang et al., 2005) and is also implicated in tumor angiogenesis (Rosen et al., 1997). The receptor for HGF, Met, is upregulated in human breast and prostate cancer patients and is known to promote metastasis (Birchmeier et al., 2003; Knudsen and Edlund, 2004). HGF stimulation of Met induces dimerization and autophosphorylation of the receptor. Autophosphorylation of tyrosine residues 1234 and 1235 in the catalytic domain is critical for Met activation (Longati et al., 1994; Rodrigues and Park, 1994). Other phosphorylated tyrosine residues, including tyrosines 1349 and 1356, serve as docking sites which recruit multiple SH2 domain containing signaling molecules to the Met receptor (Ponzetto et al., 1994). Signaling molecules recruited to Met include Grb2, PI3 kinase, Src, PLC γ, Cbl, Crk, and Gab1. Gab1 is a large adaptor molecule which is 96

110 tyrosine phosphorylated and recruited to the Met receptor following Met activation (Weidner et al., 1996). Gab1 also contains a number of tyrosine residues which, when phosphorylated, serve as docking sites for the recruitment of additional signaling molecules and are required for Met signaling (Bardelli et al., 1997; Sakkab et al., 2000). Both Crk and its family member CrkL have been shown to bind tyrosine phosphorylated YXXP motifs on Gab1 (Lamorte et al., 2002b; Sakkab et al., 2000). Crk is required for the morphological changes induced in epithelial cells in response to HGF treatment, and overexpression of Crk in MDCK cells promotes cell spreading, induction of lamellipodial protrusions, activation of Rac, and dissolution of adherens junctions, thus mimicking HGF mediated effects (Lamorte et al., 2002b). The Abl family of nonreceptor tyrosine kinases, Abl and Arg, participate in signaling cascades leading to a number of cellular responses including regulation of cell proliferation, survival, response to extracellular stresses, cytoskeletal rearrangments, and adhesion in various tissues (Pendergast, 2002). Previously, Abl kinase has been implicated in regulating signaling downstream of the PDGF and EGF growth factor receptors (Plattner et al., 1999; Tanos and Pendergast, 2006). Abl kinases activate several downstream molecules, including Crk, Rac, Cdc42, Wave, and Abi1 (Bierne et al., 2005; Bosse et al., 2007; Burton et al., 2003) which are also activated following HGF stimulation of the Met receptor (Royal et al., 2000). HGF stimulated collagen invasion of MDCK cells has been linked to several signaling molecules, including N WASP and Rac, which 97

111 operate downstream of Abl kinases in other signaling pathways (Burton et al., 2005; Burton et al., 2003; Miao et al., 2003; Yamaguchi et al., 2002). Further, Abl kinase activity is upregulated in a number of metastatic vs. non metastatic breast cancer cell lines, and has been shown to mediate invasion (Srinivasan and Plattner, 2006). These observations led us to investigate whether Abl kinases play a role in modulating the cellular response to Met activation. 4.2 Results Abl kinases function downstream of the activated Met receptor tyrosine kinase for induction of cell scattering The Abl kinase has been shown to regulate motility induced by Met activation (Abassi and Vuori, 2002; Frasca et al., 2001). To test the hypothesis that Abl kinases function downstream of HGF and its receptor, the Met tyrosine kinase to induce breakdown of cell cell junctions, we first examined whether HGF treatment of MDCK epithelial cells could induce activation of the endogenous Abl family kinases. Using phosphorylation of the Abl substrate Crk as a readout, we observed that the endogenous Abl kinases are markedly activated as early as 5 minutes after HGF stimulation (Figure 19A). HGF induced activation of the Abl kinases persists after 15 minutes of HGF stimulation. To test whether Abl kinases play a role in HGF induced morphogenesis, 98

112 A 20 ng/ml HGF untreated 5 min 15 min B IP: CrkL STI571: HGF: IB: pcrk IB: CrkL None - 15 min HGF 4 h HGF +10 µm STI min HGF 4 h HGF β-catenin actin Figure 19: Abl kinases act downstream of c Met following HGF stimulation. Figure 19: (A) MDCK cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/ml HGF. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of CrkL followed by immunoblotting with a phosphospecific antibody recognizing Y207 in CrkL, the Abl specific site of tyrosine phosphorylation. The blot was stripped and re probed for CrkL to demonstrate equivalent protein levels. (B) MDCK cells were seeded onto glass coverslips and starved for 4h. Cells were pre treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were stimulated with 20 ng/ml HGF for the indicated times and were fixed and stained for β catenin (upper panels) and actin (lower panels). In the absence of HGF stimulation, there was no difference between control and STI571 treated cells. At 15m following HGF stimulation, STI571 treated cells spread more quickly than control cells. By 4h, cell spreading was similar in both groups. In control cells, cell cell contacts dissolved in response to HGF as evidenced by relocalization of β catenin to the cytoplasm and extensive remodeling of cortical actin. However, in STI571 treated cells, β catenin remained localized to sites of cell cell contact and thick bundles of cortical actin remained in many cells. 99

113 Abl kinase activity was inhibited with STI571 prior to stimulation of MDCK epithelial cells with HGF. HGF stimulation results in morphological changes that lead to cell scattering and epithelial mesenchymal transition (EMT). Cell cell adhesion is disrupted after 4 hours of HGF stimulation (Figure 19B). In contrast, inhibition of the endogenous Abl kinases with STI571 blocks the dissolution of adherens junctions (stained with β catenin) and inhibits actin remodeling at sites of cell cell contact in the presence of HGF (Figure 19B). Similar effects were observed in A431 epithelial cells (data not shown). Inhibition of the Abl kinases specifically inhibits HGF induced morphological changes, as in the absence of HGF stimulation no difference is observed between control and STI571 treated cells. Thus, the data show that endogenous Abl tyrosine kinase activity is required for HGF induced remodeling of adherens junctions and the actin cytoskeleton. HGF stimulation of Met results in increased tyrosine phosphorylation of β catenin (Hiscox and Jiang, 1999; Monga et al., 2002). Tyrosine phosphorylation of β catenin on residues 654 and 142 disrupts its association with E cadherin and α catenin, respectively (Brembeck et al., 2004; Piedra et al., 2003; Roura et al., 1999). Phosphorylation at Y654 has been linked to disruption of adherens junctions and dissociation of the cadherin catenin complex (Piedra et al., 2001). We have shown that expression of activated Abl results in phosphorylation of β catenin at Y654 (unpublished data). We reasoned that STI571 inhibition of Abl kinase activity may block Abl mediated phosphorylation of β catenin at Y654, thereby stabilizing the link between the cadherin 100

114 catenin complex and blocking HGF induced adherens junction remodeling. However, we observed no change in levels of overall tyrosine phosphorylation of β catenin or of phosphorylated Y654 (data not shown) suggesting that Abl regulates the physiological consequence of HGF stimulation in MDCK cells through alternative pathways Abl kinase inhibition alters signaling downstream of the activated Met receptor Tyrosine phosphorylation of Crk downstream of Met activation in MDCK cells has been previously reported, and Crk was postulated to be a direct substrate of the Met receptor (Lamorte et al., 2000; Sakkab et al., 2000). Our data showing that tyrosine phosphorylation on the Abl specific site of Crk suggested that Abl may be responsible for Crk phosphorylation. To test this, we treated MDCK cells with HGF in the presence or absence of STI571 and examined phosphorylation of Crk. We observed increased tyrosine phosphorylation of Crk in HGF stimulated cells; however, phosphorylation was not induced in the presence of the Abl kinase inhibitor (Figure 20). A direct role for Abl in Crk phosphorylation is further supported by the recent finding that HGF induced phosphorylation of Crk in HeLa cells is inhibited by both STI571 treatment and expression of a dominant negative, kinase dead form of Abl (Abassi and Vuori, 2002). Abl has previously been identified in a screen for binding partners of Met (Weidner et al., 1996). Recently, our lab demonstrated that Abl associates with EGFR and 101

115 20 ng/ml HGF 0 min 5 min 15 min 30 min control +STI control +STI control +STI contol +STI IB: pcrkl Y207 IB: Crk Figure 20: Abl kinase inhibition blocks tyrosine phosphorylation of Crk in response to Met activation. Figure 20: MDCK cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/ml HGF. Cells were pre treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoblotting with a phospho specific antibody recognizing Y207 in CrkL, the Abl specific site of tyrosine phosphorylation. The blot was stripped and reprobed for CrkL to demonstrate equivalent protein levels. 102

116 directly phosphorylates tyrosine residues in the EGFR cytoplasmic tail to regulate endocytosis (Tanos and Pendergast, 2006). We hypothesized that Abl may play a similar role in Met receptor regulation. To test this, we stimulated MDCK cells with HGF in the presence or absence of STI571 and examined phosphorylation of the Met receptor using phospho specific antibodies. Autophosphorylation of tyrosine residues 1234 and 1235 in the catalytic domain is critical for Met activation (Longati et al., 1994; Rodrigues and Park, 1994). We observed significantly decreased phosphorylation of both Y1234/1235 in the presence of Abl kinase inhibition (Figure 21). This data suggests that while autophosphorylation of Y1234/1235 following dimerization of Met molecules may initiate signaling, Abl mediated transphosphorylation of Y1234/1235 may be required for sustained signaling. Phosphorylation of tyrosine 1003 of the Met receptor is required for Cbl mediated ubiquitination and internalization of Met (Peschard et al., 2004). We examined phosphorylation of Y1003 in response to HGF stimulation in the presence or absence of STI571 and observed a reduction in tyrosine phosphorylation in the presence of Abl kinase inhibition suggesting that Y1003 is another potential target of the Abl kinases (Figure 21). A Met receptor mutant in which this tyrosine has been mutated to phenylalanine (Met Y1003F) shows increased receptor stability, prolonged activation of signaling pathways, including the MAPK pathway, and enhanced transformation in response to HGF stimulation (Abella et al., 2005). We examined the effect of Abl kinase inhibition on Erk activation following HGF treatment and observed both elevated levels 103

117 20 ng/ml HGF 0 min 5 min 15 min 30 min control +STI control +STI control +STI contol +STI IB: py1003 Met IB: py1234/1235 Met IB: Met IB: perk IB: Erk IP: Gab1 IB: ptyr Figure 21: Inhibition of Abl kinase activity affects Met signaling downstream of HGF stimulation. Figure 21: MDCK cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/ml HGF. Cells were pre treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Tyrosine phosphorylation of Met was evaluated using phospho specific antibodies to Y1234/1235 and Y1003. Erk activation was assessed using a phospho specific antibody. Total protein levels for Met and Erk are indicated. Tyrosine phosphorylation of Gab1 was assessed by immunoprecipitation of Gab1 followed by immunoblotting with antibody to phosphotyrosine. 104

118 of perk as well as prolonged phosphorylation (Figure 21) similar to the effect of the Met Y1003F mutant (Abella et al., 2005). This data is consistent with the finding that perk is elevated in thyroid cancer cells treated with STI571 and HGF (Frasca et al., 2001). Thus, Abl kinases alter phosphorylation of the Met receptor in response to HGF stimulation, possibly through direct phosphorylation, and alter downstream signaling. Gab1 is a large adaptor molecule recruited to the Met receptor following Met activation, which contains 6 YXXP sites that become tyrosine phosphorylated following HGF stimulation (Weidner et al., 1996). A number of tyrosine residues, when phosphorylated, serve as docking sites for the recruitment of additional signaling molecules and play a critical role in Met signaling (Bardelli et al., 1997; Sakkab et al., 2000). The Crk adaptor protein is protein is recruited to tyrosine 307 of Gab1 and is required for sustained Gab1 phosphorylation and physiological response to HGF stimulation in synovial sarcoma cells (Watanabe et al., 2006). We and others have identified Crk phosphorylation downstream of HGF stimulation as an Abl mediated process in other cell types; therefore, we hypothesized that Abl may play a role in regulating Gab1 phosphorylation downstream of Met activation. To test this, we examined HGF stimulated phosphorylation of Gab1 in the presence or absence of STI571. We observed a nearly complete loss of Gab1 tyrosine phosphorylation in response to HGF in the presence of Abl kinase inhibition, with no change in total Gab1 suggesting that Abl kinases are required for Gab1 phosphorylation (Figure 21). Abl 105

119 mediated phosphorylation of Gab1 may occur directly or indirectly through Abl regulation of Met and/or Crk tyrosine phosphorylation in response to HGF treatment. 4.3 Discussion Here we identify the Abl family of tyrosine kinases as critical mediators of HGFstimulated dissolution of adherens junctions in epithelial cells. Pharmacological inhibition of Abl kinase activity blocks re localization of β catenin to the cytoplasm and remodeling of cortical actin in response to Met receptor activation. Further, we show that Abl kinase activity is increased in response to HGF stimulation resulting in the tyrosine phosphorylation of Crk at the Abl specific site. We observed no detectable changes in the levels of tyrosine phosphorylation of β catenin in cells treated with HGF and the Abl kinase inhibitor STI571, suggesting that the effect of Abl is not due to direct regulation of the cadherin catenin complex. We have previously shown that Abl is required for adherens junction formation (Zandy et al., 2007); thus, we have demonstrated a requirement for Abl in both dissolution and formation of adherens junctions. This is not unsurprising considering that Rac activation leading to actin polymerization and Src kinase activity have been shown to be required for both processes (Avizienyte et al., 2002; Calautti et al., 1998; Ehrlich et al., 2002; Noren et al., 2001; Royal et al., 2000). A simple explanation may involve the degree to which Abl kinase activity is activated: cadherin activation results in activation of the Abl kinases, but at a relatively low level compared to activation following HGF stimulation. Perhaps 106

120 a low basal level of Abl kinase activity is required for the maintenance of adherens junctions, but high levels of Abl kinase activity may result in the phosphorylation of different targets which play a negative role in regulating adherens junctions. In this regard, a recent report demonstrated similar requirement for Src kinase activity (McLachlan et al., 2007). Significantly, we show for the first time that Met receptor phosphorylation and activation is impaired in the absence of Abl kinase activity. Interestingly, a previous report found that STI571 treatment of thyroid cancer cells led to increased tyrosine phosphorylation of the Met receptor in response to HGF stimulation (Frasca et al., 2001). This suggests that the consequences of Abl kinase inhibition may be context dependent. While we examined immortalized normal canine kidney cells, this report used highly motile cancer cells, in which other growth factor receptor signaling pathways or downstream targets may be upregulated. Another difference may be the presence of strong adherens junctions in MDCK cells as compared to the cells examined in the other study. In this regard, Abl may act to integrate signaling from adhesion receptors, such as E cadherin, and the Met receptor with adhesion playing a role in determining the consequence of Met activation and vice versa. In addition, we showed decreased phosphorylation of Gab1, a critical regulator of Met signaling. Crk has been shown to be required for tyrosine phosphorylation of Gab1, and phosphorylation of Y307 of Gab1 was nearly eliminated in cells treated with 107

121 the Abl kinase inhibitor. Several possibilities exist to explain this result: Gab1 may be phosphorylated directly by Abl or Gab1 may be phosphorylated by the Met receptor, itself, or by another downstream kinase. In this regard, we showed that Abl kinase inhibition decreased phosphorylation of Met at the autophosphorylation site Y1234/1235, which is correlated with its activity. Alternatively, Abl mediated phosphorylation of Crk may be required for Gab1 activity. This hypothesis is supported by the finding that knockdown of Crk in synovial sarcoma cell lines led to decreased phosphorylation of Gab1 as well as impaired cell scattering in response to HGF stimulation (Watanabe et al., 2006). Our observation that Abl kinase inhibition also inhibits the cell scattering response to HGF suggests that tyrosine phosphorylation of Crk rather than the presence or absence of Crk protein determines cellular response to HGF stimulation. Our finding that Abl kinase inhibition leads to decreased phosphorylation of Met at Y1003 and increased phosphorylation of Erk in response to HGF stimulation suggests that Abl may play an additional role in signal attenuation. Cbl is recruited to Y1003 of the Met receptor phosphorylation and is required for monoubiquitination of the Met receptor, thereby promoting lysosomal degradation (Abella et al., 2005). The mutant Met receptor Y1003F displays sustained signaling, including enhanced, prolonged phosphorylation of Erk, thus mimicking the effect of Abl kinase inhibition. Future studies will be directed at evaluating the contribution of Abl kinases to the various steps 108

122 in this process and to determine whether, similar to Met Y1003F, inhibition of Abl kinase activity promotes transformation (Abella et al., 2005). In this context, Abl may participate in a negative regulatory feedback loop with the Met receptor. In agreement with this hypothesis, a recent study suggested that Abl mediated phosphorylation of Crk acts as a negative regulator of cell motility downstream of Met signaling in HeLa cells stimulated with HGF or in fibroblasts expressing the Tpr Met oncoprotein (Abassi and Vuori, 2002). Taken together, these results suggest that Abl may play both a positive and negative regulatory role in Met receptor signaling. One possible explanation for this phenomenon is that Abl kinase activation of different targets downstream of Met activation is governed through spatio temporal regulation. Alternatively, the biological consequence of Abl kinase activity may be determined by cell type. Cells with weak intercellular adhesions, which display enhanced motility in response to HGF stimulation may utilize Abl kinase activity to attenuate Met signaling. While cells with strong intercellular adhesions may require Abl kinase activity to activate and sustain Met signaling. In this regard, Abl kinases have been shown to play a role in cellular processes downstream of diverse signaling molecules including growth factor receptors, integrins, Src family kinases, and reactive oxygen species (Pendergast, 2002). Perhaps these pathways converge on Abl, whose job it is to interpret the complex message and pass it along to elicit the proper functional consequence. 109

123 5. Materials and Methods 5.1 Antibodies and Reagents Antibodies and chemical reagents used were: N cadherin, β tubulin, actin, all mouse monoclonal (Sigma), pcrkl Y207, pcrk Y221, pmlc, all rabbit polyclonal (Cell Signaling Technology), E cadherin and , β catenin, p120 catenin, p190 RhoGAP, Crk, all mouse monoclonal (BD Transduction Labs), α catenin, mouse monoclonal (Zymed), Abl K12, CrkL, Met sc 162, all rabbit polyclonal (Santa Cruz), Abl 8E9, mouse monoclonal (BD Pharmingen), Rac, Rho, both mouse monoclonal(pierce), GFP (Roche), Gab rabbit polyclonal (Upstate Biotechnology), anti py1003 Met AF4059 and anti py1234/2345 Met AF2480, both rabbit polyclonal (R & D Systems), CY3 donkey anti mouse, CY3 donkey anti rabbit, CY2 donkey anti mouse (Jackson ImmunoResearch), Alexa488 conjugated phalloidin, protein A Sepharose, protein G Sepharose, HRP linked protein A, ECL western blotting reagents (Amersham), HRPlinked goat anti mouse IgG (Santa Cruz), HRP linked goat anti rabbit IgG, DAPI (Molecular Probes), Y27632 (Calbiochem), human basic FGF B (PeproTech), mouse VEGF V4512 and human HGF H1404 (Sigma). Antibody to Arg was produced by immunizing rabbits with a peptide specific for a unique C terminal region (Finn et al., 2003). STI571 was a gift from B. Druker (Oregon Health Sciences University). The pk1 110

124 Arg GFP plasmid was obtained from A. Koleske (Yale University). pk1 ArgPP (Plattner et al., 2004), MigR1 RacV12 and MigR1 CrkYF (Burton et al., 2003) plasmids were generated as described. 5.2 Cell Lines Abl Arg double null mouse embryo fibroblasts (MEFs) were obtained from A. Koleske (Yale University) and reconstituted with Abl and Arg as previously described (Finn et al., 2003; Plattner et al., 2003). MEFs, HeLa, NBT II, MDCK, MCF 7, A431, and PY 4 1 cells were maintained in Dulbeco s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Koleske et al., 1998; Plattner et al., 2003). MCF 10A human epithelial cells were maintained in DMEM/F12 supplemented with 5% donor horse serum, hegf (20 ng/ml), hydrocortisone (500 ng/ml), insulin (10 μg/ml), and cholera toxin (100 ng/ml). 67NR, 66c14, 4TO7 and 4T1 cells were grown in DMEM supplemented with 10% FBS, 1mM non essential mixed amino acids, and 2mM L glutamine. 5.3 Retroviral Transduction Transduction of MDCK, HeLa, MCF 10A, and MEF cells was performed as described (Grove et al., 2004; Reya et al., 2003). Viral supernatant was harvested from transfected 293T cells and target cells were infected for 4 6 h. After 48h, cells were either 111

125 plated for experiments or sorted for GFP. Cells transduced with GFP Arg infected cells were subjected to FACS sorting to select a heterogeneous GFP positive population. 5.4 Cell lysis, immunoblotting, in vitro kinase assays, and RhoGTPase activation assays Cells were lysed in either Triton lysis buffer (1% Triton X 100, 150 mm NaCl, 50 mm Tris ph 7.5) or RIPA buffer (0.5 M NaCl, 1% NP 40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris ph 8.0) plus inhibitors (1mM phenylmethylsulfonyl fluoride, 2mM sodium pyrophosphate, 1 mm sodium orthovanadate, 25 mm β glycerophosphate, and 1 μg/ml each of leupeptin, aprotinin, and pepstatin A). In vitro kinase assays were performed as previously described (Plattner et al., 1999). Rac/Rho activation assays were performed according to manufacturer s instructions using the EZ Detect Rac or Rho activation kit (Pierce). Briefly, cells were lysed in lysis/binding/wash buffer plus inhibitors (1mM phenylmethylsulfonyl fluoride, Results of 3 4 independent experiments were used for quantitation of data. ImageJ Software was used to quantify relative band intensity normalized to the time 0 timepoint. Control and experimental conditions at each timepoint were compared and analyzed for statistical significance using ANOVA followed by the paired Student s T test (two tailed analysis, Excel). 112

126 5.5 Immunofluorescence Cells plated on glass coverslips were fixed with 4% (w/v) PFA for 10 min, permeabilized by the addition of 0.25% TritonX 100 for 5 min, and incubated in block (2% BSA in PBS). Antibodies were diluted in block as follows: N cadherin (1:100), E cadherin (1:200), β catenin (1:300), α catenin (1:100), 488 phalloidin (1:1000), p190 (1:200), pmlc pab (1:100), CY3 donkey anti mouse secondary (1:1000), CY3 donkey anti rabbit secondary (1:1000), CY2 donkey anti mouse secondary (1:600), DAPI (1:25,000). Fluorescence microscopy was carried out at 40x or 63x using a Zeiss Axioskop microscope equipped with a Hamamatusu ORCA ER digital camera or at 40x or 63x using a Zeiss Axiovert 200M equipped with AxioCamMRm high resolution. Images were analyzed using Metamorph software (Universal Imaging Corp.) or AxioVision 4.5 (Zeiss). Statistical analysis of adherens junction integrity in untreated vs. STI571 treated cells was performed using the paired Student s T test (two tailed analysis, Excel or Graphpad software). All other statistical analyses using multiple conditions were performed using one way ANOVA analysis followed by Bonferroni s multiple comparison test (Graphpad software). 113

127 5.6 Calcium Switch Experiments MEFs, NBT II, MCF 10A, or MDCK cells were grown to confluency, washed with PBS, and DMEM containing 10% FBS and 2mM EGTA was added overnight. The following day, medium was replaced with DMEM containing 10% FBS. For Abl kinase activity assays, NBT II cells were seeded onto glass coverslips, grown to subconfluency, washed with PBS, and medium was replaced with KBM 2 containing bovine pituitary extract (BPE), human epidermal growth factor (hegf), insulin, hydrocortisone, GA 1000, epinephrine, transferrin, and 30 μm calcium. The following day, medium was replaced with KBM 2 as described above containing 1.8 mm calcium for the indicated times. 5.7 sirna Cells were transfected with Oligofectamine (Invitrogen) and control or the indicated duplex oligonucleotides according to the manufacturer s instructions. sicontrol Non Targeting sirna Pool (D ), sigenome SMARTpool reagents human Abl1(M ), rat Abl1 (M ), human Abl2(M ), rat Abl2 (M ), and ON TARGETplus Duplex reagents human CrkL (J ), human Crk (J ), rat CrkL (J ) and rat Crk (J ) were purchased from Dharmacon. Cells were lysed 48 h following transfection. For immunostaining, siglo 114

128 RISC Free sirna (Dharmacon) was co transfected to identify cells containing control or Abl1 and Abl2 sirnas. 5.8 Growth factor stimulation MDCK or A431 cells were grown to subconfluency in 100mm plates or on glass coverslips and starved O/N in DMEM containing 0.25% FBS. Using conditioned medium, cells were pre treated or not with 10 μm STI571 for 1 h, followed by treatment with 20 ng/ml HGF for the indicated times. PY 4 1 cells were treated with 50 ng/ml VEGF, and MEFs and MCF 7 cells were treated with 20 ng/ml FGF 2 in a similar manner. 115

129 6. Conclusion Regulation of intercellular adhesion is tightly controlled during development and contributes to a number of biological processes including epithelial morphogenesis, vasculogenesis, and neural development (Thiery, 2003). Conversely, deregulation of intercellular adhesion plays a critical role during cancer progression, contributing to EMT, and promoting escape, invasion, and metastasis (Thiery, 2002). As is often the case, signaling pathways that play a role in normal development have been identified as being hijacked by tumor cells in order to grow and disseminate. The cadherin based cell adhesion complex, adherens junction, plays a critical role in development, and loss of adherens junctions has been implicated in tumorigenesis. In fact, loss of or mislocalization of members of the cadherin catenin complex has been identified in a variety of epithelial derived cancers (Christofori, 2003). Tumors may also have elevated levels of tyrosine kinase activity as well as deregulated signaling through Rho GTPases, which are two of the primary mechanisms through which adherens junctions are regulated (Steinberg and McNutt, 1999). We have described the role of the Abl family of tyrosine kinases as critical mediators of intercellular adhesion, particularly through regulation of the Rho GTPases (Zandy et al., 2007). We propose that further understanding this pathway in the context of normal and cancer cells will provide insight into potential new therapeutic interventions. 116

130 One area of adherens junction biology that is poorly understood is the mechanism through which cross talk between growth factor receptors and cadherins is regulated. Reports of interactions between EGFR and E cadherin, VEGFR and VEcadherin, and FGFR and N cadherin pepper the cadherin literature, yet the significance and generalizability of these findings remains unclear (Brunton et al., 2004). We and others have previously shown that Abl kinases are activated downstream of PDGF and EGF stimulation and play a role in cytoskeletal reorganization (Boyle et al., 2007; Plattner et al., 1999; Sini et al., 2004; Tanos and Pendergast, 2006). We have also described the role of Abl in regulating signaling downstream of cadherin molecules (Zandy et al., 2007). Therefore, the Abl kinases, acting downstream of both RTKs and cadherins, appear to be perfectly positioned to integrate signaling from the two inputs and to regulate cross talk between them. The effect of Met receptor activation on adherens junctions is probably the most well characterized RTK/cadherin interaction. Met and E cadherin have been shown to interact physically, and HGF stimulation of Met has been shown to promote relocalization of β catenin to the cytosol, thereby disrupting adherens junctions (Birchmeier et al., 2003). We have now shown that Abl kinases are required for disruption of adherens junctions downstream of Met activation and play a role in modulating Met signaling. While the mechanism through which Abl acts is at this time unclear, our data suggest that spatio temporal regulation of Abl kinase activity is 117

131 important and that the biological consequence of Abl activation is likely to be cell typeand context dependent. This novel finding raises the exciting possibility that the Abl kinases act downstream of other RTK/cadherin interactions to integrate signaling and regulate cytoskeletal changes. 6.1 Delving deeper into signaling downstream of cadherins The role of Abl kinases in regulating adherens junctions was first suggested by loss of function studies in Drosophila and recently corroborated by the finding that exogenous Abl localizes to adherens junctions in Drosophila (Grevengoed et al., 2001; Stevens et al., 2007). Our studies have confirmed that the Abl kinases play a critical role in promoting adherens junction formation and maintenance and have extend this finding to mammalian cells. We provide the first evidence of cadherin activation of Abl kinases in both fibroblasts (N cadherin) and epithelial cells (E cadherin), suggesting that the relatively conserved cytoplasmic tail of cadherins is responsible for this effect. We also showed that Abl kinases are recruited to nascent sites of cell cell contact and are found in complex with E cadherin and β catenin. At this time, the mechanism of Abl activation and recruitment to the cadherin catenin complex remains unclear. Our lab has previously shown that Src is required for activation downstream of PDGF stimulation (Plattner et al., 1999). Src has recently been shown to be activated downstream of cadherin engagement, and is postulated to function upstream of PI 3 kinase activation 118

132 (McLachlan et al., 2007). Therefore, the role of Src kinases in Abl activation downstream of cadherins should be investigated. Another avenue for future study is evaluating the requirement for individual domains in the Abl kinases. Our finding that pharmacological inhibition and sirnaknockdown of Abl and Arg result in the same phenotype suggest a critical role for the Abl kinase domain in regulating cadherin signaling, however it is unknown whether kinase activity is required for localization to sites of cell cell context or interaction with the adhesion complex. Dissecting the role of SH3 and SH2 domain mediated proteinprotein interactions as well as contributions from the F actin binding domain of the cytoplasmic tail may shed some light on this process. Re introduction of both Abl and Arg into cells lacking both Abl family members may also reveal individual roles for each. In this regard, Abl, but not Arg, was shown to rescue PLC γ-mediated increased chemotaxis in response to PDGF in Abl / Arg / cells, in spite of the fact that both kinases are activated and form complexes with members of the PDGFR signaling components (Plattner et al., 2004). To date, the bulk of evidence has suggested that tyrosine kinases directly target members of the cadherin catenin complex, particularly β catenin, to regulate adherens junctions (Brunton et al., 2004). Additionally, tyrosine phosphorylation was thought to primarily play a negative regulatory role (Brunton et al., 2004). Our studies provide evidence of positive regulation of cadherin based adhesion through a tyrosine kinase, 119

133 whose effects do not involve directly targeting the cadherin catenin complex, but rather downstream effectors. We showed that Abl regulates the activity of Rho GTPases downstream of cadherin engagement. This represents a significant contribution to understanding how signals are transduced through cadherin molecules to the Rho GTPases resulting in actin polymerization. Further, this represents another pathway in which Abl acts upstream of Rac to promote its activation. In this regard, Abl is required for Rac activation downstream of both growth factor receptor stimulation and bacterial infection (Burton et al., 2003; Sini et al., 2004). The inhibitory effect of the Abl kinases on Rho signaling has also been observed in the context of integrin engagement (Bradley et al., 2006). The mechanism through which Abl modulates Rho activity remains unclear. Abl may inhibit Rho through intermediates like p190 RhoGAP, which has been identified as a substrate for Arg (Bradley et al., 2006; Hernandez et al., 2004) or Ablmediated activation of Rac may lead to inhibition of Rho activity (Nimnual et al., 2003; Wildenberg et al., 2006). It seems likely that the Abl kinases contribute to regulation of Rac and Rho activity in other signaling cascades and should be examined Role for Abl and Crk Our studies led to the unexpected observation that Abl and Crk promote adherens junction formation and stability by acting both upstream and downstream of activated Rac. This is the first evidence of a positive feedback loop downstream of cadherin engagement, which could help explain the sustained activation of Rac during 120

134 this process. We found that Abl phosphorylates Crk in response to cadherin engagement, and that Abl activity and Crk phosphorylation are required to stimulate Rac activation in this context. Phosphorylation of Crk is required for Rac localization to the plasma membrane and activation in other contexts, however the exact mechanism through which it acts is unknown (Abassi and Vuori, 2002; Burton et al., 2003). The Rac GEF DOCK180 is an attractive candidate downstream of Crk (Kiyokawa et al., 1998). Alternatively, Abl kinases may promote Rac activation independently of Crk. In this regard, Abl mediated activation of Rac downstream of growth factor signaling requires direct phosphorylation of the Rac Gef Sos 1 (Sini et al., 2004). The significance of Crk tyrosine phosphorylation was highlighted by the finding that overexpression of Crk, which led to increased levels of unphosphorylated Crk, promoted the disruption of adhesion. This phenotype was rescued by co expression with ArgPP, which suggested that the overabundance of unphosphorylated Crk was responsible for the dissolution of adherens junctions. Therefore, evaluating the biological consequence of loss of Crk should be accompanied by studies examining its phosphorylation. Tyrosine phosphorylation of Crk has been shown to alter complex formation with other molecules, including p130 Cas, and this regulation has been implicated in regulating cell migration and focal adhesion formation (Stupack et al., 2000; Takino et al., 2003). The combination of knocking down Crk family proteins using RNAi and expressing Crk WT or the mutant Crk Y221F will allow the signaling 121

135 contributions of unphosphorylated vs. phosphorylated Crk to be dissected. Our findings also suggest the possibility that unphosphorylated Crk may play a dominant role over phosphorylated Crk as the levels of phosphorylated Crk did not decrease over baseline in cells overexpressing Crk. We also observed that expression of constitutively active RacV12 resulted in increased Abl kinase activity and Crk tyrosine phosphorylation. This data suggests that one way in which actin polymerization may be sustained throughout the hours long process of adherens junction formation is through this feedback loop. It also highlights the difficulty of studying this dynamic process. The only way to truly understand the hierarchy of signaling is to dissect the signaling pathway in a temporal manner. This could be done by examining the timing of recruitment/activation of Abl Crk Rac via live imaging. The mechanism through which Rac activation stimulates Abl activity is unknown, but may occur through Rac mediated generation of ROS. Reactive oxygen species have been shown to promote Abl kinase activation and tyrosine phosphorylation of Crk (Sun et al., 2000b). Future studies using ROS scavengers will be helpful in determining the potential significance of this pathway Abl and p190 RhoGAP Whereas p190 RhoGAP is an Arg substrate and is implicated in Arg mediated regulation of neural development and integrin engagement (Bradley et al., 2006; Hernandez et al., 2004), our studies do not support a role for p190 RhoGAP in the 122

136 regulation of adherens junctions downstream of the Abl kinases in epithelial cells or fibroblasts. p190 A RhoGAP was recently shown to function with p120 catenin in the regulation of cell cell adhesion in mouse embryo fibroblasts (MEFs) expressing constitutive active Rac V12 (Wildenberg et al., 2006). We found no evidence that p190 A (endogenous or exogenously expressed) localizes to adherens junctions in the epithelial or fibroblast cell lines we studied (Figure 22B). Thus, we expressed activated DA Rac in fibroblasts (MEFs) and did not observe detectable p190 RhoGAP localization at cell cell junctions in these cells or in MEFs expressing activated Arg, which we have shown induces strengthening of adherens junctions similar to DA Rac (data not shown). Moreover, we detected no change in junctional integrity of p190 A null MEFs and pegfp p190 A reconstituted null MEFs in the absence or presence of DA Rac or activated Arg PP (Figure 22A). However, we observed on unexplained and puzzling finding in p190 A null MEFs, which had been reconstituted with pegfp p190a: in all cells reconstituted with p190, tyrosine phosphorylation of CrkL at the Abl specific site was elevated as compared to control cells (Figure 22C). Additionally, we observed increased phosphorylation of CrkL Y207 in MEFs expressing both constitutively active Arg and Rac, further corroborating evidence observed in epithelial cells (Figure 22C). This suggests the intriguing possibility that p190 RhoGAP may play some role in the Abl Crk Rac positive feedback loop we have postulated. 123

137 A vector p190 -/- IF: β-catenin +pegfp p190 WT B vector IF: p190 RhoGAP +pegfp p190 WT NBT-II cells D DA-Rac ArgPP IP: P120 RasGAP WCL sirna: IB: p190 RhoGAP IB: p120 RasGAP IB: p190 RhoGAP IB: p120 RasGAP MCF-10A Cntrl Abl1/2 C STI571 0 h 3 h sirna: IB: pcrkl Y207 IB: Abl/Arg NBT-II Control Abl/Arg vector p190 -/- + p190 WT E DA-Rac ArgPP vector IP: P190 RhoGAP IP: P190 RhoGAP DA-Rac ArgPP ptyr p190 p120 catenin p190 NBT-II Control Abl/Arg Figure 22: Lack of evidence for role of Abl kinase modulation of p190 RhoGAP effect on adherens junctions. 124

138 Figure 22: (A) p190 RhoGAP null or pegfp p190rhogap reconstituted MEFs were obtained from Jeff Settleman (Harvard). MEFs were transduced with retroviruses encoding DA Rac, ArgPP, or vector alone, grown to near confluency and fixed and stained with antibody against β catenin (red) and counterstained with DAPI (blue) to evaluate adherens junctions. (B) pegfp p190rhogap reconstituted MEFs obtained from the Settleman lab (left panel) or NBT II epithelial cells (right panel) were fixed and stained with antibody against p190 RhoGAP (red) and counterstained with DAPI (blue) to assess p190 localization. (C) p190 null and pegfp p190rhogap reconstituted MEFs obtained from the Settleman lab expressing DA Rac, ArgPP, or vector alone were lysed in 1% Triton X 100 lysis buffer containing protease and phosphatase inhibitors. A phospho specific antibody to CrkL Y207 was used to assess levels of CrkL phosphorylation at the Abl specific site, and 8E9 was used to evaluate Abl/Arg protein expression. (D) MCF 10A cells (left panels) were transfected with control or Abl and Arg directed sirnas or were treated or left untreated with 10 μm STI571. NBT II cells (right panels) were transfected with control or Abl and Arg directed sirnas. Cells were lysed in 1% Triton X 100 lysis buffer containing protease and phosphatase inhibitors. p120 RasGAP was immunoprecipitated from 500 μg lysate, followed by immunoblotting for p190 RhoGAP. Blots were stripped and re probed for p120 RasGAP. The indicated antibodies were used to detect p120 RasGAP and p190 RhoGAP in cell lysates. (E) NBT II cells were transfected with control or Abl and Arg directed sirnas and lysed in 1% Triton X 100 lysis buffer containing protease and phosphatase inhibitors. p190 RhoGAP was immunoprecipitated from 1 mg lysate, followed by immunoblotting for phosphotyrosine or p120 catenin as indicated. Blots were stripped and re probed for p190 RhoGAP. 125

139 We also investigated whether loss of Abl kinases induced by knockdown of Abl/Arg, or treatment with the Abl kinase inhibitor STI571 affected p190 RhoGAP tyrosine phosphorylation or the formation of P190 protein complexes with either p120 RasGAP or p120 catenin. In response to integrin engagement, p190 RhoGAP is phosphorylated by Arg which promotes its binding to p120 RasGAP (Bradley et al., 2006; Hernandez et al., 2004). We did not observe any changes in tyrosine phosphorylation of p190 RhoGAP in epithelial cells lacking Abl kinases or in the formation of p190/p120 RasGAP or p190/p120 catenin complexes in response to cadherin engagement in the absence of Abl kinases compared to control cells (Figure 22, E). Thus, together our data suggest that Abl targets other than p190 RhoGAP mediate the Abldependent regulation of adherens junctions. Similarly, loss of p190 RhoGAP is dispensable for PDGF induced formation of dorsal ruffles, a process mediated by Abl kinases (Boyle et al., 2007; Plattner et al., 1999). Thus, whereas Arg and p190 RhoGAP activities are linked in response to integrin engagement, their functions appear to be uncoupled in other processes such as growth factor induced dorsal ruffle formation and cadherin dependent adherens junctions. However, the possibility exists that Abl may signaling through p190 RhoGAP regulates adherens junctions in other contexts. It is possible that the contribution of Ablmediated p190 phosphoryation was undetectable in the cells we studied, but could play a more prominent role in other cell types. For example, Src mediated phosphorylation of 126

140 p190 RhoGAP has been shown to regulate EGFR signaling (Haskell et al., 2001a; Haskell et al., 2001b). Therefore, the contribution of Src to p190 RhoGAP phosphoryation may obscure Abl mediated changes, particularly when they occur at such a specialized site as the adherens junction. Similarly, the p190 RhoGAP contribution to adherens junction regulation may be especially prominent when cadherin signaling is particularly strong. For example, induction of p190 RhoGAP phosphorylation was initially observed by directly activating cadherins using the soluble cadherin extracellular domain (Noren et al., 2003; Wildenberg et al., 2006) Abl and other downstream effectors Our data reveal a critical role for Crk phosphorylation downstream of Abl activation in regulating adherens junction formation and stability. However, there are certainly other candidate effector molecules, which may act as Abl targets downstream of cadherin signaling. In cells expressing constitutively activated ArgPP, we observed slightly elevated levels of p120 catenin at tyrosine 228 (data not shown). Phosphorylation of Y228 on p120 catenin by Src has been shown to promote binding of p120 catenin to Rho, thereby promoting Rho inhibition (Castano et al., 2007). This suggests a potential mechanism through which expression of ArgPP leads to strengthening of intercellular adhesions. Arg mediated tyrosine phosphorylation of p120 catenin could lead to enhanced binding and inhibition of Rho activity. Rho inhibition is required for the formation of cadherin mediated cell contacts, and our data 127

141 suggest that Abl normally functions to maintain low levels of Rho activity in confluent cells. We did not detect any difference in p120 catenin phosphorylation in Abl loss of function studies, suggesting that p120 catenin may only be a substrate for Abl kinases when they are overexpressed or hyperactivated. Cortactin represents another possible Abl mediated substrate downstream of cadherin engagement. Recently, Arg was shown to phosphorylate cortactin in response to PDGF stimulation, and cortactin has been shown to function downstream of E cadherin and N cadherin to promote intercellular adhesion (Boyle et al., 2007; El Sayegh et al., 2004; Helwani et al., 2004). Both loss and gain of function studies with Abl and Arg will be required to evaluate whether cortactin acts downstream of the Abl kinases to regulate adhesion. Finally, Arg was recently shown to phosphorylate myosin IIb (Boyle and Koleske, 2007). We have observed that Abl kinase inhibition leading to Rho activation promotes phosphorylation of MLC leading to enhaced acto myosin contractility, thereby disrupting adherens junctions. The biological consequences of Arg mediated phosphorylation of myosin IIb are unknown at this time, but future studies may reveal a role for this phosphorylation in adherens junction regulation. In this regard, myosin IIb was found to interact with N cadherin and β catenin in the spinal canal of mice, and genetic ablation of myosin IIb resulted in loss of intercellular adhesion (Ma et al., 2007). 128

142 Our studies have revealed a significant novel role for the Abl kinases in the positive regulation of adherens junctions. We have identified Crk as a direct Abl substrate, which is required for adherens junction formation and strengthening. However, gaps in our knowledge regarding the mechanism of Abl kinase activation and the identity of other potential Abl targets remain. 6.2 Dissecting the role of Abl downstream of Met receptor activation Data from Chapter 4 reveal a novel role for the Abl kinases in mediating the disruption of adherens junctions downstream of Met activation. We add Met to a list of tyrosine kinase growth factor receptors, including PDGFR and EGFR, which activate Abl kinases (Plattner et al., 1999; Sini et al., 2004). Previous studies have similarly shown that Abl kinase activity is upregulated following HGF stimulation, and the Abl kinase has been shown to negatively regulate motility induced by Met activation (Abassi and Vuori, 2002; Frasca et al., 2001). In contrast, our findings suggest a positive role for the Abl kinases in regulating Met signaling. We hypothesize that differences in cell type, level and duration of Abl kinase activation, inputs from other signaling pathways, and different downstream effectors play a role in the Abl dependent regulation of Met signaling. 129

143 6.2.1 Abl and Met One of our most interesting findings is that Abl kinase inhibition results in decreased phosphorylation of the Met autophosphorylation sites Y1234/1235. While this may be an indirect effect, we propose that Abl directly phosphorylates the Met receptor and contributes to a positive feedback loop, which contributes to sustained signaling. Abl was previously identified in a screen for binding partners of Met (Weidner et al., 1996), and binding may be required to allow Abl to directly phosphorylate Met or may induce a conformational change which sustains Met kinase activity. Our lab has recently shown that Abl kinases can directly phosphorylate the EGFR, and that phosphorylation leads to sustained signaling and decreased endocytosis (Tanos and Pendergast, 2006). In the case of EGFR, this leads to hyperactivation of signaling pathways (Tanos and Pendergast, 2006). However, HGF stimulation of the Met receptor is known to lead to sustained signaling as compared to stimulation of other growth factor receptors, therefore it is possible that aberrant signaling in the case of EGFR is similar to normal signaling by Met. It is also unknown whether Met directly phosphorylates Abl leading to its activation or whether other signaling molecules may modulate the effects of Met activation on Abl. In this regard, PLC γ is recruited to the Met receptor following HGF stimulation and promotes Abl activation downstream of PDGF stimulation (Plattner et al., 2004). Future studies will be directed at furthering an understanding of the interaction between Abl and Met in this system. Expression of kinase dead mutants of 130

144 Abl and Met in the presence of wild type Met and Abl, respectively, will shed light on whether Abl directly phosphorylates Met in response to HGF stimulation and vice versa. Additional studies using GFP tagged Abl or Arg in combination with live cell imaging can be used to assess whether Abl kinases are, as we expect, recruited to the Met receptor following HGF stimulation, and allow us to evaluate the duration of this interaction. Additionally, we will use biochemical means to evaluate Met Abl binding. Kinase dead mutants will also be useful to determine whether kinase activation is required for binding. The Met receptor contains multiple tyrosines, which when phosphorylated allow SH2 domain containing proteins to bind (Ponzetto et al., 1994). The use of both tyrosine to phenylalanine mutant Met receptors and Abl kinases lacking functional domains, such as SH2 may allow the binding regions on both proteins to be identified. Further, the contribution of phosphorylation sites and specific domains to recruitment to the plasma membrane and phosphorylation and activation of both Abl and Met could be determined. Phosphorylation of tyrosine 1003 of the Met receptor is required for Cblmediated ubiquitination and internalization of Met (Peschard et al., 2004). We observed reduced phosphorylation of Y1003 in response to HGF stimulation in the presence of Abl kinase inhibition suggesting that Y1003 is another potential target of the Abl kinases. This finding is particularly interesting because mutations in this region of the Met receptor have been implicated in tumorigenesis, therefore suggesting a role for the 131

145 Abl kinases in these tumors (Kong Beltran et al., 2006). In vitro data support a critical role for Y1003 in cancer. A Met receptor mutant in which this tyrosine has been mutated to phenylalanine (Met Y1003F) shows increased receptor stability, prolonged activation of signaling pathways, and enhanced transformation in response to HGF stimulation (Abella et al., 2005). Met Y1003F fails to bind Cbl, which prevents its trafficking to the lysosome for degradation (Abella et al., 2005). Further, Met Y1003F results in sustained signaling through the MAPK pathway and increased and prolonged phosphorylation of Erk. Our data also showed that Abl kinase inhibition decreased phosphorylation of Met at Y1003 and increased phosphorylation of Erk. We hypothesize that Abl phosphorylates the Met receptor downstream of HGF stimulation and normally acts to promote turnover of Met. We expect that inhibition of Abl kinases will phenocopy expression of Met Y1003F, and future studies will examine this hypothesis. We expect that loss of Abl kinases will result in increased stability of the Met receptor, and expect to see enhanced, prolonged cytoplasmic staining of both Met and Erk. We also expect Abl kinase inhibition or knockdown of Abl/Arg to prevent binding of Cbl with Met and the subsequent monoubiquitination of the Met receptor Abl and other downstream targets Tyrosine phosphorylation of Crk in response to HGF stimulation was nearly eliminated in cells treated with the Abl kinase inhibitor. Previously, Crk has been shown to be required for disruption of adherens junctions following Met activation, and Crk 132

146 overexpression leads to increased Rac activation and cell spreading in MDCK cells in the absence of Met activation (Lamorte et al., 2002a; Lamorte et al., 2002b). This effect was similar to what we observed in response to STI571 inhibition of Abl kinase activity and Crk overexpression in NBT II cells, thus suggesting that unphosphorylated Crk is responsible for these biological effects. Crk phosphorylation in response to HGF stimulation, is in fact, transient suggesting that the timing and duration of Crk phosphorylation may be critical for Met signaling. For example, early signaling events downstream of Met activation may require phosphorylation of Crk. In this regard, tyrosine phosphorylation of Crk has been shown to be required for Rac recruitment to the plasma membrane, and this may be the case in early Met signaling (Abassi and Vuori, 2002). That overexpression of Crk resulted in enhanced cell spreading (Lamorte et al., 2002b) and inhibition of Abl kinase activity enhances motility in response to HGF (Cipres et al., 2007; Frasca et al., 2001) suggests a negative role for Abl Crk in modulating Met signaling. These responses are not immediate, and unphosphorylated Crk may be required for late signaling events downstream of Met activation. Future studies which carefully examine the phosphorylation status of Crk localization and its effects on Rac activity as well as phenotypic consequences will be required to reevaluate the role of Crk phosphorylation. Alternatively, the different effects of Crk may be dependent on other factors such as cell type or strength of intercellular adhesion. We have demonstrated that the Abl Crk Rac signaling module acts downstream of cadherin 133

147 engagement, therefore, the contribution of cadherin signaling may alter signaling downstream of Met activation. Gab1 is a large adaptor molecule recruited to the Met receptor following Met activation. Gab1 contains 6 YXXP sites that become tyrosine phosphorylated following HGF stimulation and serve as docking sites for the recruitment of additional SH2 domain containing proteins to the Met signaling complex (Weidner et al., 1996). The Crk adaptor protein is recruited to phosphorylated tyrosine 307 of Gab1 and is required for sustained Gab1 phosphorylation and physiological response to HGF stimulation in synovial sarcoma cells (Watanabe et al., 2006). Our studies reveal a role for Abl kinases in modulating Gab1 phosphorylation, and the numerous YXXP sites in Gab1 make it yet another candidate substrate for Abl downstream of Met activation. If Abl is not recruited directly to Met, Gab1 also represents a likely intermediate which might recruit Abl to allow it to participate in Met signaling. Supporting this possibility, BcrAbl has been shown to associate with the closely related Gab2 protein in chronic myelogenous leukemia lines (Samanta et al., 2006). We examined total phosphorylation of Gab1, but use of phosphospecific antibodies or tyrosine to phenylalanine mutants of Gab1 may reveal which tyrosines are phosphorylated by Abl directly or indirectly. It is also unclear at this time whether Abl directly phosphorylates Gab or whether Gab1 phosphorylation is affected indirectly through Abl mediated effects on the Met receptor or Crk. Future studies will be directed at examining the functional consequences of loss of Abl kinases 134

148 with regard to Gab binding to Met and recruitment of SH2 domain containing proteins, including Crk. If Abl is found to directly phosphorylate specific sites, studies using Gab1 tyrosine to phenylalanine mutants could be use to examine the biological consequences of Abl loss of function on Gab1 signaling. Abl may also function to regulate interactions between β catenin and E cadherin downstream of Met activation. HGF stimulation of Met results in increased tyrosine phosphorylation of β catenin (Hiscox and Jiang, 1999; Monga et al., 2002). Phosphorylation at Y654 has been linked to disruption of adherens junctions and dissociation of the cadherin catenin complex (Piedra et al., 2001). While we have observed no change in tyrosine phosphorylation of β catenin in response to HGF stimulation in our studies, that does not eliminate the possibility that in some contexts Abl phosphorylation of β catenin regulates cell scattering downstream of Met. Another possible explanation of how Abl kinase inhibition blocks adherens junction turnover is that endocytosis and turnover of the Met receptor and E cadherin are somehow linked. If Abl kinase inhibition mimics the effect of increased Met Y1003F stability, and Met is not degraded, it is possible that E cadherin recycling may also be blocked. However, rather than remaining in the cytoplasm, E cadherin may be recycled back to the plasma membrane. Very little is known regarding the mechanisms through which the Met receptor regulates adherens junctions, therefore highlighting the significance of our finding that Abl kinases act downstream of both Met receptor and cadherins. 135

149 While our preliminary findings support a critical role for the Abl kinases in regulating Met signaling, the Met signaling cascade is highly complex and contains many candidate Abl kinase substrates to study. Once a clear understanding of how the Abl kinases affect the response to HGF stimulation in one cell type, it will be important to examine the generalizability to other cell types. For cells in which Abl appears to act differently, factors such as cell type, cell confluence, and presence or absence of other signals which stimulate Abl kinase activity, including other growth factor receptors and cadherins will need to be examined. Additionally, while we have examined disruption of adherens junctions in response to Met, we have not examined other physiological responses to HGF such as motility, invasion, transformation, angiogenesis, or tumorigenesis (Birchmeier et al., 2003). Our finding that Abl kinases play a role in adherens junction remodeling in response to HGF have opened the door to studying the potential role for Abl kinases as master regulators integral for interpreting signaling inputs and allowing cross talk between growth factor receptors and cadherins. 6.3 Role of Abl downstream of other growth factors Our lab and others have shown that the Abl kinases are activated downstream of various growth factors and participate in signaling downstream of PDGFR and EGFR (Boyle et al., 2007; Plattner et al., 1999; Sini et al., 2004; Tanos and Pendergast, 2006). Our data from Chapter 5 suggest that the Abl kinases function as critical intermediaries in modulating communication between the activated Met receptor and the cadherin 136

150 catenin complex. While HGF induced disruption of adherens junctions is perhaps the most well characterized and well understood example of cross talk between RTKs and the cadherin based adhesion complex, other growth factor receptors have been implicated in adherens junction remodeling. For example, our lab has observed a role for the Abl kinases in modulating endocytosis of the EGFR, whereby expression of constitutively active AblPP blocks EGF stimulated endocytosis (Tanos and Pendergast, 2006). EGF stimulation of the EGFR has been shown to increase caveolin mediated endocytosis of E cadherin (Lu et al., 2003). We have observed that expression of ArgPP results in strengthening of N cadherin based cell adhesion in HeLa cells (Zandy et al., 2007). Taken together, these data suggest the possibility that cross talk from the EGFR to cadherins may be responsible for this phenotype if E cadherin endocytosis is dependent on endocytosis of the EGFR and suggests an area for future study Abl and FGF N cadherin and FGFR have been shown to act cooperatively to promote FGF2 stimulated signaling, resulting in increased invasion and angiogenesis (Derycke et al., 2006; Suyama et al., 2002). Abl kinases have been implicated in negatively regulating N cadherin based adhesion in neurons by mediating cross talk between Robo and N cadherin (Rhee et al., 2007; Rhee et al., 2002). We propose that the Abl kinases act as master regulators at the intersection of RTK and cadherin signaling, thus we expect the 137

151 Abl kinases may play a role in modulating FGFR/N cadherin cross talk in epithelial cells. We have previously established a role for Abl kinases in regulating N cadherinbased intercellular adhesion (Zandy et al., 2007). To evaluate the possibility that Abl acts downstream of the FGFR as well, we stimulated WT or Abl / Arg / MEFs with FGF2 and examined phosphorylation of Crk at the Abl specific site. We observed increased Abl kinase activity by 5 minutes in WT cells, with no phosphorylation of Crk in cells lacking Abl kinases (Figure 23A). To test whether this pathway functions in epithelial cells, we stimulated MCF 7 cells with FGF2, and again observed elevated levels of Abl kinase activity 15 minutes following stimulation (Figure 23B). Taken together, these data support our hypothesis that Abl kinases are perfectly positioned to integrate signaling between the FGFR and N cadherin. Further, many of the same signaling pathways in which Abl has been implicated play a critical role in modulating FGF2 signaling, including Erk, Grb2, and PLCγ. Future experiments will be directed at uncovering which physiological processes are mediated by Abl kinases (proliferation, migration, invasion, and/or angiogenesis) and dissecting the role of Abl kinases in the FGF2 stimulated signaling pathway. We surmise that Abl may play a role in these processes and possibly also in cadherin switching during tumorigenesis in epithelial cells. An alternative, and equally exciting, possibility is that Abl kinases may also modulate angiogenesis through mediating FGF2 signaling in endothelial cells. Our novel finding that Abl kinases are 138

152 A 20 ng/ml FGF2 0 min 5 min 10 min 20 min null Abl/Arg null IP: Crk Abl/Arg null Abl/Arg null Abl/Arg IB: pcrk IB: Crk B 0 min 5 min 10 min IP: Crk 20 min IB: pcrk IB: Crk Figure 23: FGF2 stimulation activates endogenous Abl kinases. Figure 23: (A) Abl/Arg null MEFs transduced with retroviruses encoding Abl and Arg or vector alone cells were were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/ml FGF2. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of Crk followed by immunoblotting with a phospho specific antibody recognizing Y221 in Crk, the Abl specific site of tyrosine phosphorylation. The blot was stripped and re probed for Crk to demonstrate equivalent protein levels. (B) MCF 7 cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/ml FGF2 in the presence or absence of 10 μm STI571. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of Crk followed by immunoblotting with a phospho specific antibody recognizing Y221 in Crk, the Abl specific site of tyrosine phosphorylation. The blot was stripped and re probed for Crk to demonstrate equivalent protein levels. 139

153 activated by FGF2 opens the door for studies of Abl s involvement in yet another pathway critically involved in tumorigenesis and tumor progression Abl and VEGF VEGF stimulation induces multiple effects in endothelial cells, including increased proliferation and vessel development. However, VEGF stimulation also can increase vascular permeability. One of the ways in which VEGF stimulation may promote vessel leakiness is by disrupting VE cadherin based intercellular adhesion. In this regard, VEGF stimulation of VEGFR has been shown to increase tyrosine phosphorylation of members of the cadherin based adhesion complex in endothelial cells, including VE cadherin, β catenin, and p120 catenin (Esser et al., 1998). Tyrosine phosphorylation of both E cadherin and β catenin have been shown to disrupt intercellular adhesion in other cell types (Brembeck et al., 2004; Fujita et al., 2002; Piedra et al., 2001; Roura et al., 1999). Thus, VEGF induced tyrosine phosphorylation of adherens junction components may lead to disruption of cadherin based adhesion, thereby promoting vascular permeability. We identified a role for the Abl kinases in positively regulating intercellular adhesion in fibroblasts in epithelial cells (Zandy et al., 2007), but have also observed a negative role for Abl kinases downstream of Met activation. These observations led us to hypothesize that Abl kinases may likewise play both a positive role downstream of 140

154 cadherin engagement and a negative role downstream of growth factor stimulation in regulating VE cadherin based intercellular adhesion in endothelial cells. To examine whether Abl kinase activity is required for the formation of adherens junctions in endothelial cells, we performed a calcium switch to induce cadherin engagement in the presence or absence of STI571 inhibition of Abl kinase activity (Figure 24). We observed reformation of adherens junctions by 2 h post switch in control cells; however, β catenin remained cytosolic in cells treated with the Abl kinase inhibitor (Figure 24A). This supports a positive role for Abl kinases in promoting VE cadherin based intercellular adhesion. To examine the other side of our hypothesis, we first needed to determine whether Abl was activated by VEGF stimulation. Using phosphorylation of Crk at the Abl specific site as a readout, we examined Abl kinase activation in response to VEGF stimulation in PY 4 1 cells and observed elevated levels of Abl kinase activity at 5 and 15 min (Figure 24B). Together, these data place the Abl kinases in a position to modulate the negative effects of VEGF stimulation on adherens junctions. In fact, VEGF stimulation of PY 4 1 cells resulted in relatively rapid re localization of β catenin from sites of cell cell contact to the cytoplasm. In contrast, β catenin remained at sites of cellcell contact in cells treated with the Abl kinase inhibitor STI571 supporting the hypothesis that Abl kinases might act to promote disruption of endothelial adherens junctions downstream of VEGF stimulation. The signaling events which govern VEGFinduced vascular permeability remain poorly understood, but likely involve many of the 141

155 A Hi Calcium Low Calcium 30 min +calcium 2 h +calcium - 5 min15 min 50 ng/ml VEGF : - 5 min 15 min B 50 ng/ml VEGF : IB: pcrkl Y207 IB: CrkL IP: CrkL control IB: CrkL (WCL) + 10 µm STI µm STI571 control C Figure 24: Abl kinases mediate intercellular adhesion in endothelial cells. Figure 24: (A) PY 4 1 cells were grown to subconfluency and subjected to calcium switch to stimulate adherens junction formation in the presence or absence of 10 μm STI571. Cells were fixed and stained with antibody against β catenin (red) and counterstained with DAPI (blue). (B) PY 4 1 cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 50 ng/ml VEGF. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of CrkL followed by immunoblotting with a phosphospecific antibody recognizing Y207 in CrkL, the Abl specific site of tyrosine phosphorylation. The blot was stripped and re probed for CrkL to demonstrate equivalent protein levels. (C) PY 4 1 cells were seeded onto glass coverslips and starved for 4h. Cells were pre treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were stimulated with 50 ng/ml VEGF for the indicated times and were fixed and stained for β catenin. 142